BRPI0707944B1 - gene construct, vector, transgenic microorganism, and processes for converting 18: 2 * 9,12 (linoleic acid) to 20: 2 * 11,14 - Google Patents

Abstract

nucleic acid. The present invention relates to perkinsus marinus-derived nucleic acid encoding a 9-elongase enzyme, a Î ”8-desaturase and a β-5 desaturase. All coding sequences can be transcribed as a single transcript.

Description

Report of the Invention Patent for "GENE CONSTRUCTION, VECTOR, TRANSGENIC MICRORGANISM AND PROCESSES FOR THE CONVERSION OF 18: 2Δ912 (LINLEIC ACID) IN 20: 2Δ11'14". The present invention relates to Perkinsus marinus-derived nucleic acid encoding a 9-elongase, A8-desaturase and an A5-desaturase enzyme. All coding sequences can be transcribed as a single transcript, which simplifies the cell transformation process required to express all three proteins. The invention also relates to the individual coding sequences and proteins encoded by these sequences as well as a process for converting linoleic acid to arachidonic acid.

Fatty acids and triglycerides have a multitude of applications in the food industry, animal nutrition, cosmetics and the pharmaceutical sector. Depending on whether they are saturated or unsaturated fatty acids or triacylglycerides with a high saturated or unsaturated fatty acid content, they are suitable for many different applications. Polyunsaturated fatty acids such as linoleic acid and linolenic acid are essential for mammals as they cannot be produced by them. The ω3 fatty acids and polyunsaturated ω6 fatty acids are therefore an important component in animal and human nutrition. Hereinafter, the polyunsaturated fatty acids are referred to as PUFA, PUFAs, LCPUFA or LCPUFAs (polyunsaturated fatty acids, PUFA, long chain polyunsaturated fatty acids, LCPUFA).

The various fatty acids and triglycerides are mainly obtained from microorganisms such as Mortierella and Schizochytrium or from oil producing plants such as soybean, col-za seed oil, algae such as Crypthecodinium or Phaeodactylum and others where they are obtained, such as a rule in the form of its triglycerides Follows sheet 1 to (= triglycerides = triglycerols). However, they can also be obtained from animals, such as, for example, fish. Free fatty acids are advantageously prepared by hydrolysis. Long chain polyunsaturated fatty acids such as docosae-xanoic acid (= DHA, C22: 6_4,7,10,13,16,19), eicosapentaenoic acid (= EPA, C20: 5A5'8'η14'17) , arachidonic acid (= ARA, C20: 4A5'8'11 · 14), dihomo-y-linolenic acid (C20: 3A8, 11, 14) or docosapentaenoic acid (DPA, C22: 5A7'10'13, 16,19), are not synthesized in oil crops such as rapeseed, soybean, sunflower or safflower oil. Conventional natural sources of these fatty acids are fish, such as herring, salmon, sardines, red fish, eel, carp, trout, halibut, mackerel, zander or tuna, or algae.

Depending on the intended use, oils with saturated or unsaturated fatty acids are preferred. In human nutrition, for example, lipids with unsaturated fatty acids, specifically polyunsaturated fatty acids, are preferred. The u) 3-polyunsaturated fatty acids are said to have a positive effect on blood cholesterol level and thus on the possibility of preventing heart disease. The risk of heart disease, stroke or hypertension can be reduced notably by adding these u) 3-fatty acids to food. In addition, ω3-fatty acids have a positive effect on inflammatory processes, specifically chronically inflammatory processes, in association with immune diseases such as rheumatic arthritis. They are then added to edibles, specifically dietary edibles, or employed in medicines. Fatty acids u> 6 such as arachidonic acid tend to have a negative effect on these disorders with respect to these rheumatic diseases because of the usual dietary influx. Ω3 and ω6 fatty acids are precursors of tissue hormones known as eicosanoids, such as prostaglandins which are derived from dihomo-hinolic acid, arachidonic acid and eicosapentaenoic acid, and thromboxanes and leukotrienes that are derived from arachidonic acid. and eicosapentaenoic acid. Eicosanoids (known as the PG2 series) which are generally formed of Î ± 6-fatty acids promote inflammatory reactions, while eicosanoids (known as the PG3 series) of ω3 fatty acids have little or no pro-inflammatory effect. inflammatory.

Due to the positive characteristics of polyunsaturated fatty acids, there has been no shortage of attempts in the past to manufacture available genes that are involved in the synthesis of these fatty acids or triglycerides for the production of oils in various organisms with a modified unsaturated fatty acid content. . Accordingly, WO 91/13972 and its equivalent U.S. describe an A9 desaturase. WO 93/1 1245 claims an A15 desaturase and WO 94/11516 an Δ12 desaturase. For example, additional desaturases are described in EP-A-0 550 162, WO 94/18337, WO 97/30582, WO 97/21340, WO 95/18222, EP-A-0 794 250, Stukey et al., J. Biol. Chem., 265, 1990: 20144-20149, Wa-da et al., Nature 347, 1990: 200-203 or Huang et al., Lipids 34, 1999: 649-659. However, the biochemical characterization of the various desaturases has been insufficient to date as enzymes, being membrane-bound proteins, have great difficulty in their isolation and characterization (McKeon et al., Methods in Enzymol. 71, 1981: 1214112147, Wang and others, Plante Physiol., Biochem., 26, 1988: 777-792). As a rule, membrane-bound desaturases are characterized by being introduced into a suitable organism which is subsequently analyzed for enzyme activity by analyzing the starting materials and products. Δ6-Desaturases are described in WO 93/06712, US 5,614,393, US5614393, WO 96/21022, WO 00/21557 and WO 99/27111 and the application for the production of fatty acids in transgenic organisms is described in WO 98 / 46763, WO 98/46764 and WO 98/46765. In this context, the expression of various desaturases and the formation of polyunsaturated fatty acids is also described and claimed in WO 99/64616 or WO 98/46776. With respect to the expression efficacy of desaturases and their effect on the formation of polyunsaturated fatty acids, it should be noted that expression of a single desaturase as described to date has only resulted in low unsaturated fatty acid / lipid content such as, for example, acid. γ-linolenic and stearidonic acid. In addition, a mixture of fatty acids u> 3 and ω6 was obtained as a rule.

Microorganisms especially suitable for the production of PUFAs are microalgae such as Phaeodactylum tricornutum, Porphiridium, Thraustochytrium species, Schizochytrium species or Crypthecodinium species, ciliates such as Stylonychia or Colpidium, fungi such as Mortierella, Entomophthora or Mucus mosses such as Physcomitrella, Ceratodon and Marchantia (R. Vazhappilly & F. Chen (1998) Botanica Marina 41: 553-558; K. Totani & K. Oba (1987) Lipids 22: 1060-1062; M. Akimoto et al. ( 1998) Appl, Biochemistry and Biotechnology 73: 269-278). Strain selection has resulted in the development of various mutant strains of the microorganisms in question that produce a number of desirable compounds including PUFAs. However, mutation and selection of strains with improved production of a particular molecule such as polyunsaturated fatty acids is a lengthy and difficult process. This is why recombinant methods as described above are preferred whenever possible.

However, only limited amounts of the desired polyunsaturated fatty acids such as DPA, EPA or ARA can be produced with the help of the aforementioned microorganisms, and, depending on the microorganism used, these are generally obtained as mixtures of fatty acids from, for example, EPA. , DPA and ARA.

A variety of synthetic reaction series are being described by the synthesis of arachidonic acid, eicosapentaenoic acid (EPA) and docosaexanoic acid (DHA) (Figure 1). Thus, EPA or DHA is produced in marine bacteria such as Vibrio sp. or Shewanella sp. by the polyketide reaction series (Yu, R., et al., Lipids 35: 1061-1064, 2000; Takeyama, H., et al., Microbiology 143: 2725-2731,1997).

An alternative strategy is the alternate activity of desaturases and elongases (Zank, T.K. et al., Plant Journal 31: 255-268, 2002; Saku-radani, E., et al., Gene 238: 445-453, 1999). A modification of the reaction series described above by A6-desaturase, A6-elongase, A5-desaturase, A5-elongase and A4 desaturase is the Sprecher reaction series (Sprecher 2000, Biochim. Biophys. Acta 1486: 219-231) in mammals. Instead of Δ4-desaturation, another stretching step is performed here to produce C2A, followed by another A6-desaturation and finally β-oxidation to produce the C22 chain length. Thus, what is known as the Sprecher reaction series (see figure 1) is, however, not suitable for production in plants and microorganisms since regulatory mechanisms are not known.

Depending on their desaturation pattern, polyunsaturated fatty acids can be divided into two large classes, viz. Fatty acids ω6 or ω3, which differ with respect to their metabolic and functional activities (Figure 1). The starting material for the w6 metabolic reaction series is fatty acid linoleic acid (18: 2Δ9,12) while the ω3 reaction series proceeds through linolenic acid (18: 3Δ9,12,15). Linolenic acid is formed by the activity of a ω3 desaturase (Tocher et al. 1998, Prog. Lipid Res. 37, 73-117; Domergue et al. 2002, Eur. J. Bio-chem. 269, 4105-4113).

Mammals, and thus also humans, have no corresponding desaturase activity (Δ12 - and Δ oo3 desaturase) and must absorb these fatty acids (essential fatty acids) by food. Starting with these precursors, the physiologically important polyunsaturated fatty acids arachidonic acid (= ARA, 20: 4Δ5,8,11,14), a ω6 fatty acid and the two ω3 fatty acid eicosapentaenoic acids (= ΕΡΑ, 20: 5δ5'8 '11' h 17) and docosaexanoic acid (DHA, 22: 6Δ4'7'10'13'17'19) are synthesized by the sequence of desaturase and elongase reactions. Application of ω3 fatty acids shows the therapeutic activity described above in the treatment of cardiovascular disease (Shimikawa 2001, World Rev. Nutr. Diet. 88, 100-108), Entzündungen (Calder 2002, Proc. Nutr. Soc. 61, 345358) and Arthritis (Cleland and James 2000, J., Rheumatol. 27, 2305-2307). The elongation of fatty acids by elongases through 2 or 4 C atoms is of crucial importance for the production of C20 - and C22-PUFAS, respectively. This process proceeds through 4 steps. The first step is the condensation of malonyl-CoA with fatty acid-acyl-CoA through ketoacyl-CoA synthase (KCS, hereinafter called elongase). This is followed by a reduction step (ketoacyl-CoA reductase, KCR), a dehydration step (dehydratase) and a final reduction step (enoyl-CoA reductase). It has been postulated that elongase activity affects the specificity and rate of the entire process (Millar and Kunst, 1997 Plant Journal 12: 121-131).

There have been a large number of attempts in the past to obtain elongase genes. Millar and Kunst, 1997 (Plant Journal 12: 121-131) and Millar et al., 1999, (Plant Cell 11: 825-838) describe the characterization of plant elon gases for the synthesis of monounsaturated long chain fatty acids. (C22: 1) and for the synthesis of very long chain fatty acids for waxing in plants (C28-C32). Descriptions regarding the synthesis of arachidonic acid and EPA are found, for example, in WO0159128, W00012720, W002077213 and W00208401. The synthesis of C24-polyunsaturated fatty acids is described, for example, in Tvrdik et al. 2000, JCB 149: 707-717 or W00244320.

Taller plants include polyunsaturated fatty acids such as linoleic acid (18: 2Δ9,12) and linolenic acid (18: 3Δ9,12,15). ARA, EPA and DHA are not found in the seed oil of taller plants, or only in small quantities (E. Ucciani: Nouveau Dictionnaire des Huiles Vegetales). Technique & Documentation - Lavoisier, 1995. ISBN: 2-7430-0009-0). However, the production of LCPUFAs in higher plants, preferably in oil crops such as rapeseed, flaxseed, sunflower and soybean oil would be advantageous since large quantities of high quality LCPUFAs for the food industry, animal nutrition and pharmaceutical purposes could be obtained economically. To this end, it is advantageous to introduce genes encoding LCPUFA biosynthesis enzymes into oil crops by recombinant methods and to express them herein. These genes may encode, for example, A9-elongases, Δδ-desaturases and / or Δδ-desaturases. These genes may advantageously be isolated from lower microorganisms and plants that produce LCPUFAs and incorporate them into membranes or triglycerides. Thus, it was already possible to isolate the Δ6 desaturase genes from the Physcomitrella patens moss and P. patens A6-elongase genes and the C. elegans nematode.

The first transgenic plants that understand and express the genes encoding LCPUFA biosynthesis enzymes and which, as a consequence, produce LCPUFAs were first described, for example, in DE-A-102 19 203 (process for the production of polyunsaturated fatty acids in plants). However, these plants produce LCPUFAs in quantities that require further optimization to process the oils that are present in the plants.

As can be seen from Figure 1, the ωΔΘ-series reaction products can be modified using appropriate desaturases and, if necessary, linkages / igases to produce ω3 fatty acids. Therefore, it would be extremely valuable to develop a product that makes ARA production possible in a genetically modified organism. The oyster protozoan parasite Perkinsus marinusi is capable of synthesizing saturated and unsaturated fatty acids, including essential fatty acid, arachidonic acid [20: 4 (n - 6), by the Δ -8 desaturase reaction series. Surprisingly, the present inventors found that P. marinusi contains nucleic encoding an A9-elongase, an Δ8-desaturase and a Δ5-desaturase which can all be transcribed as a single transcript. The life-size sequence is shown as SEQ ID NO: 1.

Thus, in a first aspect of the invention, an isolated nucleic acid sequence encoding A9-elongase, Δδ desaturase and Δ5 desaturase activity polypeptides is selected from the group consisting of: a) Nucleic acid sequence comprising nucleic acid residues 7668 to 12077 of SEQ ID NO: 1 or a homologue thereof; (b) a nucleic acid sequence hybridizing under stringent conditions to a nucleic acid sequence comprising nucleic acid residues 7668 to 12077 of SEQ ID NO: 1; c) an isolated nucleic acid sequence encoding the polypeptides with A9-elongase, Δδ-desaturase and Δδ-desaturase activity, wherein the polypeptides are selected from the group consisting of SEQ ID NOS 2, 3 and 4; d) A derivative of a nucleic acid sequence of SEQ ID NO: 1 encoding polypeptides of at least 40% amino acid identity with SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4; wherein said polypeptides have Δθ-elongase, Δ8 - desatu-rase and Δδ-desaturase activity. The advantage of the nucleic acid sequence of the present invention is that although it encodes three separate enzymes, it can be transcribed as a single sequence that makes it much simpler to prepare the cloning and expression vectors that express all three enzymes.

Preferably, the isolated nucleic acid sequence according to the invention is not identical to SEQ ID NO 1 (sequence 1047306867) itself.

In the context of the present invention "hybridizing under stringent conditions" is intended to describe the hybridization and washing conditions under which nucleotide sequences with at least 60% homology to one another normally remain hybridized to one another. The conditions are preferably such that sequences of at least about 65%, preferably at least about 70% and especially preferably at least 75% or more homology to one another will normally remain hybridized to one another. These stringent conditions are known to the skilled worker and are described, for example, in Currents Protocols in Molecular Biology, John Wiley & Sons, N., Y. (1989), 6.3.1-6.3.6. A preferred non-limiting example of stringent hybridization conditions is 6x hybridization of sodium citrate / sodium chloride (= SSC) at approximately 45 ° C, followed by one or more 0.2 x SSC washing steps, 0.1% SDS at 50 to 65 ° C. The skilled worker knows that these hybridization conditions differ depending on the type of nucleic acid and, for example, when organic solvents are present, relative to temperature and buffer concentration. Under "standard hybridization conditions", for example, the hybridization temperature is, depending on the type of nucleic acid, between 42 ° C and 58 ° C in an aqueous buffer with a concentration of 0.1 to 5 x SSC (pH 7, 2). If organic solvents, for example 50% formamide, are present in the above buffer, the temperature under standard conditions is approximately 42 ° C. Hybridization conditions for DNA: DNA hybrids, for example, are 0.1 x SSC and 20 ° C to 45 ° C, preferably 30 ° C to 45 ° C. Hybridization conditions for DNA: RNA hybrids are, for example, 0.1 x SSC and 30 ° C to 550 ° C, preferably 45 ° C to 55 ° C. The aforementioned hybridization conditions are determined by way of example for a nucleic acid approximately 100 bp (= base pairs) in length and with a G + C content of 50% in the absence of formamide. The skilled worker knows how to determine the hybridization conditions required on the basis of the aforementioned textbooks or textbooks such as Sam-brook and others, "Molecular Cloning", Cold Spring Harbor Laboratory, 1989; Hames and Higgins (Ed.) 1985, "Nucleic Acids Hybridization: A Practical Appro-ach", IRL Press at Oxford University Press, Oxford; Brown (Ed.) 1991, "Essential Molecular Biology: A Practical Approach", IRL Press at Oxford University Press, Oxford.

In addition, when the present report relates to nucleic acid molecules isolated from a nucleotide sequence that hybridizes to one of the nucleotide sequences shown in SEQ ID NO: 1, or part thereof under stringent conditions. "a part thereof" is understood to mean according to the invention that at least 25 base pairs (= bp), 50 bp, 75 bp, 100 bp, 125 bp or 150 bp, preferably at least 175 bp, 200 bp, 225 bp, 250 bp, 275 bp or 300 bp, especially preferably 350 bp, 400 bp, 450 bp, 500 bp or more base pairs are used for hybridization.

In the context of the present invention "Nucleic acid sequence homologues" having the sequence SEQ ID NO: 1 means, for example, allelic variants of at least about 50 or 60%, preferably at least about 60 or 70%, more preferably at least about 70 or 80%, 90% or 95% and even more preferably at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99% or more of identity or homology to a nucleotide sequence shown in SEQ ID NO: 1. "Allelic variants" include, in particular, functional variants which may be obtained by deletion, insertion or substitution of nu-cleotides from / in sequence, however, it is intended that the enzyme activity of the resulting proteins that are synthesized be advantageously retained for insertion of one or more genes. "Homologs" also means bacterial, fungal and plant homologs, truncated sequences, single stranded DNA or RNA from the coding and non-coding DNA sequence, and derivatives such as, for example, promoter variants. Promoters upstream of the detailed nucleotide sequences may be modified by one or more nucleotide exchanges by deletion insert (s) without the functionality or activity of the adversely affected promoters, however. It is furthermore possible that modification of the promoter sequence increases its activity or that it is completely replaced by more active promoters, including those from heterologous organisms.

To determine the percent homology (= identity) of two amino acid sequences, the sequences are written under one another for optimal comparison (for example, openings in the sequence of a protein or nucleic acid can be introduced to generate alignment). ideal with the other protein or other nucleic acid). Then, the amino acid residue or nucleotide at the corresponding amino acid positions or nucleotide positions is compared. If a position in one sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the other sequence, then the molecules are homologous to that position (ie amino acid or nucleic acid "homology" as used in the present context). corresponds to the "identity" of the amino acid or nucleic acid). The percentage of homology between the two sequences is a function of the number of positions the sequences share (ie,% of homology = number of identical positions / total number of positions x 100). The terms homology and identity will then be considered as synonyms. Homology was calculated over the sequence region of nucleic acid or total amino acid. The skilled worker has available a number of programs that are based on various algorithms for comparing various sequences. Here, the algorithms of Needleman and Wunsch or Smith and Waterman give particularly reliable results. The Pil-eUp program (J. Mol. Evolution, 25, 351-360, 1987, Higgins et al., CABIOS, 5 1989: 151-153) or the Gap and BestFit programs [Needleman and Wunsch (J. Mol. Biol 48; 443-453 (1970) and Smith and Waterman (Adv. Appl. Math. 2; 482489 (1981)], which are part of the GCG software package [Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711 (1991)], was used for sequence alignment.The sequence Ogy values which are given above as a percentage were determined over the total sequence region using the GAP program the following settings: GAP Weight: 50, Weight Length: 3, Average Balance: 10,000, and Average Imbalance: 0,000.Unless otherwise specified, these settings have always been used as default settings for sequence alignments.

In the context of the present invention "A9-elongase, Δδ-desaturase and A5-desaturase activity" is understood to mean that a protein encoded by a derivative of SEQ ID NO: 1 or nucleic acid residues 7668 to 12077 of SEQ ID NO: 1 retains an enzyme activity of at least 10%, preferably 20%, especially preferably 30% and most especially 40% compared to proteins / enzymes encoded by the sequence SEQ ID NO: 1 or nucleic acid residues 7668 to 12077 of SEQ ID NO. : 1 and ttttt can thus catalyze the conversion of linoleic acid to arachidonic acid.

Although it is often extremely useful to transcribe nucleic acid encoding A9-elongase, Δδ-desaturase and A5-desaturase activity polypeptides as a single sequence, there may be some circumstances in which it is preferable to make use of nucleic acid encoding a single enzyme, that is, an A9-elongase, an Δ8-desaturase or an A5-desaturase.

Thus, in a second aspect of the invention there is provided an isolated nucleic acid sequence encoding an A9-elongase activity polypeptide and selected from the group consisting of: a) a sequence comprising nucleic acid residues 7668 to 9200 of SEQ ID NO: 1, SEQ ID NO: 9 or a homolog of one of these; (b) nucleic acid sequences that hybridize under stringent conditions to a nucleic acid sequence comprising residues 7668 to 9200 of SEQ ID NO: 1 or SEQ ID NO: 9; c) an isolated nucleic acid sequence encoding A9-elongase activity polypeptides, wherein the polypeptide comprises SEQ ID NO: 2 or SEQ ID NO: 10; d) a derivative of a sequence comprising nucleic acid residues 7668 to 9200 of SEQ ID NO: 1 or SEQ ID NO: 9, encoding a polypeptide of at least 40% identity at the amino acid level of SEQ ID NO: 2 or SEQ ID NO: 10; wherein said polypeptide has A9-elongase activity.

In a third aspect of the invention there is provided an isolated nucleic acid sequence encoding a Δ8-desaturase activity polypeptide selected from the group consisting of: a) a sequence comprising nucleic acid residues 9351 to 10724 of SEQ ID NO: 1 or a homologue thereof; (b) nucleic acid sequences hybridizing under stringent conditions to a nucleic acid sequence comprising residues 9351 to 10724 of SEQ ID NO: 1; c) an isolated nucleic acid sequence encoding A8-desaturase activity polypeptides, wherein the polypeptide comprises SEQ ID NO: 3;

d) a derivative of a sequence comprising nucleic acid residues 9351 to 10724 of SEQ ID NO: 1 encoding a polypeptide with at least 40% amino acid level identity to SEQ ID NO: 3; wherein said polypeptide has Δδ desaturase activity.

In a fourth aspect of the invention there is provided an isolated nucleic acid sequence encoding a Δ5 desaturase activity polypeptide selected from the group consisting of: a) a sequence comprising nucleic acid residues 10842 to 12077 of SEQ ID NO: 1 or a homologue thereof; (b) nucleic acid sequences hybridizing under stringent conditions to a nucleic acid sequence comprising residues 10842 to 12077 of SEQ ID NO: 1; c) an isolated nucleic acid sequence encoding polypeptides with Δδ desaturase activity, wherein the polypeptide comprises SEQ ID NO: 4; d) a derivative of a sequence comprising nucleic acid residues 10842 to 12077 of SEQ ID NO: 1 encoding a polypeptide of at least 40% identity at the amino acid level of SEQ ID NO: 4; wherein said polypeptide has Δδ desaturase activity.

In yet another aspect of the invention, there is provided a polypeptide which is encoded by a nucleic acid sequence of any of the first to fourth aspects of the invention.

Advantageously, the polypeptide encoded by these nucleic acid molecules is at least about 60%, preferably at least about 60% and more preferably at least about 70%, 80% or 90% and preferably at least about 8δ%, 86%. 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 9δ%, 96%, 97%, 98%, 99% or more of identity with the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 9.

The nucleic acid sequences used in the process are advantageously introduced into an expression cassette which makes the expression of nucleic acids possible in organisms such as microorganisms or plants.

Then, in another aspect of the invention there is provided a gene construct comprising a nucleic acid sequence encoding one or more A9-elongase, Δδ-desaturase and / or A5-desaturase activity polypeptides as shown above, operably linked with one or more more regulatory sequences.

In the expression cassette, the nucleic acid sequence encoding A9-elongase, Δδ-desaturase and / or Δδ-desaturase is operably joined with one or more regulatory sequences, advantageously to enhance gene expression. These regulatory sequences are intended to make specific expression of genes and proteins possible. Depending on the host organism, this may mean, for example, that the gene is expressed and / or overexpressed only after induction has occurred, or that it is expressed and / or overexpressed immediately. For example, these regulatory sequences take the form of sequences to which inducers or repressors bind, thereby controlling the expression of nucleic acid. In addition to these new regulatory sequences, or instead of these sequences, the natural regulatory elements of these sequences may still be present before the current structural genes and, if appropriate, may have been genetically modified in such a way that their natural regulation is eliminated and expression of genes is intensified. However, the expression cassette (= expression construct = gene construct) may also be simpler in the construct, that is, no additional regulatory signal was inserted before the nucleic acid sequence or derivatives thereof, and the natural promoter along with its regulation has not been removed. Instead, the natural regulatory sequence has been mutated in such a way that regulation no longer occurs and / or gene expression is intensified. These modified promoters may also be positioned within themselves prior to the natural gene in the form of sequence parts (= promoter with parts of nucleic acid sequences used in accordance with the invention) to enhance activity. In addition, the gene construct may also advantageously comprise one or more of what are known as enhancer sequences in operable linkage with the promoter, which makes enhanced expression of the nucleic acid sequence possible.

Additional advantageous sequences, such as other regulatory elements or terminator sequences, may also be inserted at the 3 'end of the DNA sequences. One or more enzyme coding sequences that catalyze the conversion of ARA into an ω3-unsaturated fatty acid such as EPA or DHA may also be present. Thus, for example, sequences encoding an A5-elongase, Δ3-desaturase and / or A4-desaturase may be present in one or more copies of the expression cassette (= gene construct). Preferably, only one copy of the genes is present in each expression cassette. This gene construct or gene constructs may be expressed together in the host organism. In this context, the gene construct (s) may be inserted into one or more vectors and are present in the cell in free form, or may be inserted into the genome It is advantageous for the insertion of other genes into the genome when the genes to be expressed are present together in a gene construct.

In this context, regulatory factors or sequences may, as described above, preferably have a positive effect on gene expression of the introduced genes, thereby enhancing. Thus, enhancement of regulatory elements, advantageously at the transcriptional level, may occur using strong transcriptional signals as enhancers and / or promoters. In addition, however, enhanced translation is also possible, for example by improving mRNA stability.

In particular, regulatory sequences include plant sequences such as promoter and terminator sequences. The constructs may advantageously be stably propagated in microorganisms, in particular E. coli and Agrobacterium tumefaciens, under selective conditions, and make it possible to transfer heterologous DNA into plants or microorganisms.

Useful regulatory sequences are present, for example, in promoters such as the cos, tac, trp, tet, trp-tet, Ipp, lac, Ipp-lac, laclq, 17, T5, T3, gal, trc, ara, SP6, λ-PR or λ-PL and are advantageously employed in Gram-negative bacteria. Other advantageous regulatory sequences are, for example, present in the amy and SP02 Gram-positive promoters, ADC1, MFa, CA, P-60, CYC1, GAPDH, TEF, rp28, ADH or plant promoters. CaMV / 35S [Franck et al., Cell 21 (1980) 285-294], PRP1 [Ward et al., Plant. Mol. Biol. 22 (1993)], SSU, OCS, ibib, usp, STLS1, B33, in or on the ubiquitin or phaseolin promoter. Advantageous in this context are also inducible promoters such as those described in EP-A-0 388 186 (benzenesulfonamide inducible), Plant J. 2, 1992: 397-404 (Gatz et al., Tetracycline inducible), EP- A-0 335 528 (abscissic acid inducible) or promoters of WO 93/21334 (ethanol or cyclohexenol inducible). Other suitable plant promoters are the cytosolic FBPase promoter or potato ST-LSI promoter (Stockhaus et al., EMBO J. 8, 1989, 2445), glycine max phosphoribosyl pyrophosphate amidotransferase promoter (Genbank Accession No. U87999). ) or the node-specific promoter described in EP-A-0 249 676.

Especially advantageous promoters are promoters that make possible expression in tissues that are involved in fatty acid biosynthesis. Most especially advantageous are seed specific promoters such as the USP promoter as described, but also other promoters such as LeB4, DC3, phaseolin promoter or napin. Other especially advantageous promoters are seed specific promoters which may be used for monocotyledonous or dicotyledonous plants and which are described in US 5,608,152 (rapeseed oil napin promoter), WO 98/45461 (Arabidopsis oleosin promoter), US 5,504,200 (Phaseolus vulgaris phaseolin promoter), WO 91/13980 (Brassica Bce4 promoter), by Baeumlein et al., Plant J., 2, 2, 1992: 233-239 (one legume LeB4 promoter), these promoters that are suitable for dichotyledones. Examples of promoters that are suitable for monocotyledones are barley lpt-2 or lpt-1 promoter (WO 95/15389 and WO 95/23230), barley protein promoter and other suitable promoters described in WO 99/16890. .

In principle, it is possible to use all natural promoters in conjunction with their regulatory sequences, such as those mentioned above. It is also possible and advantageous to use synthetic promoters, either in addition or alone, particularly when they mediate seed specific expression, such as those described in WO 99/16890.

To achieve a particularly high ARA content, especially in transgenic plants, genes should advantageously be expressed in oil crops in a specific seed manner. For this purpose, seed-specific promoters or promoters that are active in the embryo and / or endosperm may be used. In principle, seed specific promoters can be isolated from either dicotyledonous or monocotyledonous plants. Preferred promoters are listed below: USP (= unknown seed protein) and viciline (Vicia faba) [Baumlein et al., Mol. Gen Genet, 1991, 225 (3)], napin (rapeseed oil) [US 5,608,152], acyl carrier protein (rapeseed oil) [US 5,315,001 and WO 92/18634], oleosin (Arabidopsis thaliana) [WO 98/45461 and WO 93/20216], phaseolin (Phaseolus vulgaris) [US 5,504,200], Bce4 [WO 91/13980], B4 Legumes (LegB4 Promoter) [Baumlein et al., Plant J., 2,2, 1992], Lpt2 and Ipt1 (Barley) [WO 95/15389 and WO95 / 23230], rice, corn and wheat seed specific promoters [WO 99/16890], Amy32b, Amy 6-6 and Aleurain [US 5,677,474], Bce4 (rapeseed oil) [US 5,530,149 ], glycine (soy) [EP 571 741], phosphoenol pyruvate carboxylase (soy) [JP 06/62870], ADR12-2 (soy) [WO 98/08962], isocitrate lyse (rapeseed oil) [US 5,689 .040] or α-amylase (barley) [EP 781 849]. Plant gene expression can also be facilitated by a chemically inducible promoter (see, review in Gatz 1997, Annu. Rev. Plant Physiol. Plant Mol. Biol., 48: 89-108). Chemically inducible promoters are particularly suitable when gene expression is desired to occur in a specific time frame. Examples of such promoters are a salicylic acid inducible promoter (WO 95/19443), a tetracycline inducible promoter (Gatz et al. (1992) Plant J. 2, 397- 404) and an ethanol inducible promoter.

To ensure stable integration of biosynthesis genes in the transgenic plant over a plurality of generations, it is normally necessary for each nucleic acid to encode a protein of interest to be expressed under the control of a separate promoter, preferably a promoter that differs from the others. promoters, since repeat sequence motifs can lead to T-DNA instability or recombination events. This is one reason why the nucleic acid of the present invention is particularly advantageous since sequences encoding A9-elongase, Δδ-desaturase and Δδ-desaturase can be transcribed as a single unit that needs only one promoter. Of course, it will be necessary for other genes to encode, for example, Δ5-elongase, u> 3 desaturase and / or A4 desaturase to be under the control of separate promoters.

In this context, the expression cassette is advantageously constructed such that a promoter is followed by a dividing site, advantageously in a polylinker, for insertion of the nucleic acid to be expressed and, if appropriate, a terminator sequence is positioned behind the polyligante. This sequence is repeated several times, preferably three, four or five times, so that up to five genes can be combined into one construct and introduced into the transgenic plant to be expressed. Advantageously, the sequence is repeated up to three times. To express nucleic acid sequences, the latter is inserted behind the promoter by a suitable dividing site, for example, into the polylinker. Advantageously, each nucleic acid sequence has its own promoter and, if appropriate, its own terminator sequence. Such advantageous constructs are described, for example, in DE 101 02 337 or DE 101 02 338. However, it is also possible to insert a plurality of nucleic acid sequences behind a promoter and, if appropriate, before a terminator sequence. Here, the insertion site, or sequence, of nucleic acids inserted into the expression cassette is not of critical importance, meaning that a nucleic acid sequence can be inserted at the first or last position in the cassette without its expression being substantially influenced by that. mode. Advantageously, different promoters such as, for example, the USP, LegB4 or DC3 promoter, and different terminator sequences may be used in the expression cassette. However, it is also possible to use only one type of promoter in the cassette. However, this can lead to unwanted recombination events.

As described above, the transcription of the genes that were introduced should advantageously be terminated by suitable terminator sequences at the 3 'end of the biosynthesis genes that were introduced (behind the disruption codon). An example of a sequence that can be used in this context is the OCS 1 terminator sequence. As is the case with promoters, different terminator sequences should be used for each gene. The gene construct of the present invention may also comprise fatty acid biosynthesis or lipid metabolism genes selected from the group of acyl-CoA dehydrogenase (s), acyl-ACP [= acyl-carrying protein] desaturase (s), thioesterase ( fatty acid acyl-ACP acyltransferase (s), acyl-CoA: lysophospholipid acyltransferase (s), fatty acid synthases, fatty acid hydroxylases, acetyl coenzyme A carboxylase (s), acyl coenzyme A oxidase (s) ), fatty acid desaturase (s), fatty acid acetylene ses, lipoxygenases, triacilglycerol lipases, alenoxide synthases, hydroperoxide liases or fatty acid elongase (s) such as A4 desaturase, A5 desaturase, Δ6 desaturase , Δδ desaturase, A9 desaturase, Δ12 desaturase or Δδ elongase.

These additional genes or nucleic acids can be cloned into expression cassettes, which are then used to transform plants with the help of vectors such as Agrobacterium.

Here, regulatory factors or sequences may, as described above, preferably have a positive effect, and thereby enhance, on the expression genes that have been introduced. Thus enhancement of regulatory elements may advantageously occur at the transcriptional level using strong transcription signals as enhancers and / or promoters. However, enhanced translation is also possible, for example by improving mRNA stability. In principle, expression cassettes can be used directly for plant introduction or can be introduced into a vector.

Then, in yet another aspect of the invention, there is provided a vector comprising a nucleic acid or gene construct in any of the aspects of the invention described above.

In one embodiment, the vector may be a cloning vector.

The nucleic acid sequences of the invention may be introduced alone, or preferably in combination with an expression cassette (nucleic acid construct) into an organism. To introduce nucleic acids, nucleic acids are advantageously extended and ligated in known manner. Preferably, a procedure following the protocol for Pfu DNA polymerase or a mixture of Pfu / Taq DNA polymerase is followed. Primers are selected taking into consideration the sequence to be extended. The primers should be advantageously chosen such that the amplifier comprises the total codogenic sequence from the start codon to the stop codon. After amplification, the amplifier is conveniently analyzed. For example, a gel electrophoretic separation may be performed, which is followed by a quantitative and qualitative analysis. After this, the amplified can be purified following a standard protocol (eg Qiagen). An aliquot of the purified amplifier is then available for the subsequent cloning step.

Suitable cloning vectors are generally known to the skilled worker. These include, in particular, vectors that are capable of replication in microbial systems, namely vectors which ensure efficient cloning into yeast or fungi and which make stable plant transformation possible. Those that should be mentioned in particular are various binary and cointegrated vector systems that are suitable for T-DNA mediated transformation. Such vector systems are, as a rule, characterized by the fact that they comprise the genes come required for Agrobacterium-mediated transformation and the T-DNA boundary sequences (T-DNA border). These vector systems advantageously also comprise other cis-regulatory regions such as promoters and terminator sequences and / or selection markers, by which suitably transformed organisms can be identified. While in the case of co-integrated vector systems, vir genes and T-DNA sequences are arranged in the same vector, binary systems are based on at least two vectors, one of which carries vir genes, but no T-DNA, while a second carries the T-DNA, but no gene comes. Due to this fact, the last vectors mentioned are relatively small, easy to manipulate and replicate in both Coli and Agrobacterium. These binary vectors include pBIB-HYG, pPZP, pBecks, pGreen series vectors. In accordance with the invention, Bin19, pB1101, pBinAR, pGPTV and pCAMBIA are preferably used. An evaluation of binary vectors and their use is found in Hellens et al., Trends in Plant Science (2000) 5, 446 - 451. To prepare vectors, vectors can first be linearized with restriction endonuclease (s) and then modified. enzymatically in a suitable manner. After that, the vector is purified, and an aliquot is employed for the cloning step. In the cloning step, the enzymatically cleaved and, if appropriate, purified amplified cloned vector fragments were prepared in a similar manner using ligase. In this context, a particular nucleic acid construct, or vector or plasmid construct, may have one or more than one codogenic gene segment. The codogenic gene segments in these constructs are preferably operably linked with regulatory sequences. In particular, regulatory sequences include plant sequences such as promoters and terminator sequences described above. The construct may advantageously be stably propagated in microorganisms, in particular E. coli and Agrobacterium tumefaciens, under selective conditions and makes it possible to transfer heterologous DNA into plants or microorganisms.

The nucleic acids of the invention may be introduced into organisms, such as microorganisms or advantageously plants, advantageously using cloning vectors, and thereby be used in transformation of plants such as those published and cited in: Plant Molecular Biology and Biotechnology (CRC) Press, Boca Raton, Florida), Chapter 6/7, p. 71-119 (1993); F.F. White, Vectors for Gene Transfer in Higher Plants; in: Transgenic Plants, Vol. 1, Engineering and Utilization, Ed .: Kung and R. Wu, Academic Press, 1993, 15-38; B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, Ed .: Kung and R. Wu, Academic Press (1993), 128-143; Potrykus, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991), 205-225. Thus, the inventive nucleic acids, nucleic acids and nucleic acid constructs and / or vectors used in the process can be used for recombinant modification of a broad spectrum of organisms, advantageously plants, so that the latter is make ARA the best and / or most efficient producer.

A number of mechanisms exist whereby modification of A9-elongase, A8-desaturase and Δδ-desaturase proteins is possible, so that yield, production and / or production efficiency of ARA in one plant, preferably in one plant. oil harvesting or a microorganism can be directly influenced due to these modified proteins. The number or activity of the proteins or genes may be increased so that larger amounts of gene products and ultimately greater amounts of the compounds of general formula I are produced. A de novo synthesis in an organism that needed activity and ability to biosynthesize compounds before introduction of the corresponding gene (s) is also possible. This applies analogously to combination with other desaturases or elongases or other enzymes of lipid and fatty acid metabolism. The use of various divergent sequences, that is, sequences that differ at the DNA sequence level, may also be advantageous in this context, or the use of promoters for gene expression that makes different gene expression possible over time , for example, as a function of the degree of maturity of a seed or oil storage tissue.

Due to the introduction of a gene encoding A9-elongase, Δ8-desaturase and / or A5-desaturase in an organism alone or in combination with other genes in a cell, it is not only possible to increase the biosynthesis flow to the end product. but also increase or create the corresponding triacylglycerol composition again. Likewise, the number or activity of other genes that are involved in the importation of nutrients that are required for biosynthesis of one or more fatty acids, oils, neutral and / or polar lipids may be increased, so that the concentration of these precursors, such as Factors or intermediates within the cells or within the storage compartment are thereby increased, the ability of cells to produce ARA as described below is enhanced as well. By optimizing activity or increasing the number of one or more genes encoding Δθ-elongase, Δδ desaturase and / or Δ5 desaturase that are involved in biosynthesis ARA, or by destroying the activity of one or more genes. which are involved in ARA degradation, an increased yield, production and / or efficiency of fatty acid and lipid molecule production in organisms, advantageously in plants, is made possible.

Nucleic acids, which may advantageously be used in the process are derived from bacteria, fungi, diatoms, animals such as Caenorhabditis or Oncorhynchus or plants such as algae or moss, such as the genera Shewanella, Physcomitrella, Thraustochytrium, Fusari-one, Phytophthora , Ceratodon, Mantonieila, Ostreococcus, Isochrysis, Aleuri-ta, Muscarioides, Mortierella, Borago, Phaeodactylum, Crypthecodinium, specifically of the genera and species Oncorhynchus mykiss, Xenopus lae-vis, Ciona intestinalis, Thalassiosira Pseudeoccuscus, Pseudococcus Tauri, Euglena gracilis, Physcomitrella patens, Phytophtora infestans, Fusarium graminaeum, Cryptocodinium cohnii, Wax. todon purpureus, Isochrysis galbana, Aleurita farinosa, Thraustochytrium sp., Muscarioides Viallii, Mortierella alpina, Borago officinalis, Phaeodactylum tricornutum, Caenorhabditis elegans or especially advantageously of Oncorhynchus mykiss pseudius thiiiiiiiiiiiiiiiiiiiiiiii.

In an alternative embodiment, the vector may be an expression vector designed to transform an organism into which nucleic acid must be expressed and linoleic acid converted to ARA.

These advantageous vectors, preferably expression vectors, comprise nucleic acid (s) encoding A9-elongase, Δ8-desaturase and / or Δδ-desaturase and which are described in the first to fourth aspects. of the invention.

As used herein, the term "vector" refers to a nucleic acid molecule that is capable of carrying another nucleic acid to which it is attached. One type of vector is a "plasmid", a circular double stranded DNA loop in which additional DNA segments can be ligated. An additional type of vector is a viral vector, and it is possible for additional DNA segments to be linked in the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they have been introduced (for example, bacterial vectors of bacterial replication origin). Other vectors are advantageously integrated into the genome of a host cell when they are introduced into the host cell, and thus replicate together with the host genome. In addition, certain vectors may administer the expression of genes to which they are operably linked. These vectors are referred to in the present context as "expression vectors". Typically, expression vectors are suitable for plasmid DNA recombination techniques.

In the present description where the term "plasmid" is used, it should be understood that plasmids may be substituted for other types of expression vectors, such as viral vectors, which perform similar functions. In addition, the term "vector" is also intended. comprise other vectors with which the skilled worker is familiar, such as phages, viruses such as SV40, CMV, TMV, transposons, IS elements, phasases, phagemids, cosmids, linear or circular DNA.

Advantageously recombinant expression vectors used in the process comprise the nucleic acids described below or the gene construct described above in a form that is suitable for expressing the nucleic acids used in a host cell, meaning that the recombinant expression vectors comprise one or more regulatory sequences, selected on the basis of the host cells used for expression, whose regulatory sequence (s) is operably linked to the nucleic acid sequence to be expressed. In a recombinantly expressed expression vector, "operably linked" means that the nucleotide sequence of interest is linked to the regulatory sequence (s) in such a way that expression of the nucleotide sequence is possible, and they are linked one to the other. another such that both sequences perform the predicted function that is assigned to sequence (for example, in an in vitro transcription / translation system, or in a host cell if the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). For example, these regulatory sequences are described in Goeddel: Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990), or see: Gruber and Crosby in: Methods in Plant Molecular Biology and Biotechnolgy, CRC Press , Boca Raton, Florida, Ed .: Glick and Thompson, Chapter 7, 89-108, including references cited herein. Regulatory sequences include those that administer constitutive expression of a nucleotide sequence in many host cell types and those that administer direct expression of the nucleotide sequence only in specific host cells under specific conditions. The skilled worker knows that the expression vector design may depend on factors such as the choice of host cell to be transformed, the desired expression level of the protein and the like.

The recombinant expression vectors used may be designed for the expression of Δ9 - elongase, Δδ desaturase and / or Δ5 desaturase in prokaryotic or eukaryotic cells. This is advantageous since intermediate steps of the vector construct are often performed on microorganisms due to simplicity. For example, the Δθ-elongase, Δδ-desaturase and / or Δδ-desaturase gene may be expressed in bacterial cells, insect cells (using Baculovirus expression vectors), yeast and other fungal cells (see Romans, Μ., A., et al. (1992) "Foreign gene expression in yeast: a review," Yeast 8: 423-488; van den Hondel, CAM.JJ, et al. (1991) "Heterologous gene expression in filamentous fungi", in: More Gene Manipulations in Fungi, JW Bennet & LL Lasure, Ed., Pp. 396-428: Academic Press: San Diego; and van den Hondel, CAMJJ, & Punt, PJ (1991) "Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, Peberdy, JF, et al., Ed., pp. 1-28, Cambridge University Press: Cam-bridge), algae (Falciatore et al., 1999, Marine Biotechnology.1, 3: 239-251), the ciliatos of the types Holotrichia, Peritrichia, Spirotrichia, Suctoria, Te-trahymena, Paramecium, Colpidium, Glaucoma, Platyophrya, Potomacus, De ssaturaseudocohnilembus, Euplotes, Engelmaniella and Stylonychia, in particular of the genus Stylonychia lemnae, using vectors in a transformation method as described in WO 98/01572 and preferably in multicellular plant cells (see Schmidt, R. and Willmitzer, L. (1988) "Hi-gh efficiency Agrobacterium tumefaciens-mediated transformation of Arabi-dopsis thaliana leaf and cotyledon explants" Plant Cell Rep.:583-586; Plant Molecular Biology and Biotechnology, C Press, Boca Raton, Florida, Chapter 6/7, pp.71-1 19 (1993); F.F. White, B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, Ed .: Kung and R. Wu, Academic Press (1993), 128-43; Potrykus, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991), 205-225 (and references cited herein)). Suitable host cells are also described in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). As an alternative, the recombinant expression vector may be transcribed in vitro translated, for example, using the T7 promoter and T7 polymerase regulatory sequences.

In most cases, protein expression in prokaryotes involves the use of vectors comprising constitutive or inducible promoters that administer expression of fusion or non-fusion proteins. Typical fusion expression vectors are, inter alia, pGEX (Pharmacia Biotech Inc; Smith, DB, and Johnson, KS (1988) Gene 67: 31-40), pMAL (New England Biolabs, Beverly, MA) und pRIT5 ( Pharmacia, Piscataway, NJ), where glutathione S-transferase (GST), maltose-E binding protein and protein A, respectively, are fused to the recombinant target protein.

Examples of suitable inducible non-fusion E coli expression vectors are, inter alia, pTrc (Amann et al. (1988) gene 69: 301-315) and pET 11 d (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 6089). Target gene expression of the pTrc vector is based on the transcription of a hybrid trp-lac fusion promoter by the host RNA polymerase. Target gene expression of the pET 11d vector is based on the transcription of a T7-gn10-lac fusion promoter that is mediated by a viral RNA polymerase (T7gn1) that is co-expressed. This viral polymerase is provided by the BL21 (DE3) or HMS174 (DE3) host strains of a resident λ-phage that house a T7 gn1 gene under transcriptional control of the lacUV 5 promoter.

Other vectors that are suitable for prokaryotic organisms are known to the skilled worker, such vectors are, for example, in E. coli pLG338, pACYC184, the pBR series, such as pBR322, the pUC series such as pUC18 or pUC19, series M113mp, pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24, pLG200, pUR290, plN-111113-B1, Agt11 or pBdCI, in Streptomyces plJ101, plJ364, plJ702 or plJ361, in Bacillus pUB14 pB144 or CJ141 pSA77 or pAJ667.

In another embodiment, the expression vector is a yeast expression vector. Examples for vectors for expression in S.cerevisiae yeast include pYeDessaturased (Baldari et al. (1987) Embo J. 6: 229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30: 933-943), pJRY88 (Schultz and others (1987) Gene 54: 113-123) and pYES2 (Invitrogen Corporation, San Diego, CA). Vectors and processes for constructing vectors that are suitable for use in other fungi, such as filamentous fungi, include those described in detail in: van den Hondel, CAMJJ, & Punt, PJ (1991) "Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of fungi, JF Peberdy et al., Ed., pp. 1-28, Cambridge University Press: Cam-bridge, or in: More Gene Manipulations in Fungi [JW Bennet & LL Lasuer, Ed., Pp. 396-428: Academic Press: San Diego] Other suitable yeast vectors are, for example, pAG-1, YEp6, YEpl 3 or pEMBLYe23.

As an alternative, A9-elongase, Δδ desaturase and / or Δ5 desaturase may be expressed in insect cells using Baculovirus vectors. Baculovirus expression vectors that are available for protein expression in cultured insect cells (e.g., Sf9 cells) comprise the pAc series (Smith et al. (1983) Mol. Cell Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170: 31-39).

The vectors mentioned above are only a small assessment of the appropriate vectors that are possible. Other plasmids are known to skilled workers and are described, for example, in: Cloning Vectors (Ed. Pouwels, P. H., et al., Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018). For other expression systems suitable for prokaryotic and eukaryotic cells, see Chapters 16 and 17 in Sambrook, J., Fritsch, EF, and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

In an additional embodiment of the process, A9-elongase, Δδ desaturase and / or Δδ desaturase may be expressed in unicellular plant cells (such as algae), see Falciatore et al., 1999, Marine Biotechnology 1 (3): 239-251 and references cited herein, and in higher plant plant cells (e.g., spermatophytes, such as cultivable crops). Examples of plant expression vectors include those described in detail: Becker, D., Kemper, E., Schell, J., and Masterson, R., (1992) "New plant binary vectors with selectable markers located proximally". to the left border ", Plant Mol. Biol. 20: 1195-1197; and Bevan, M.W. (1984) "Binary Agrobacterium vectors for plant transformation", Nucl. Acids Res. 12: 8711-8721; Vectors for Gene Transfer in Higher Plants; in: Transgenic Plants, Vol. 1, Engineering and Utilization, Ed .: Kung and R. Wu, Academic Press, 1993, pp. 15-38.

A plant expression cassette preferably comprises regulatory sequences that are capable of administering gene expression in plant cells and are operably linked so that each sequence can fulfill its function, such as transcriptional termination, for example polyadenylation signals. Preferred polyadenylation signals are those derived from Agrobacterium tumefaciens T-DNA, such as Ti pTiACH5 gene gene 3 (Gielen et al., EMBO J. 3 (1984) 835 et. Seq), which is known as octopine synthase. , or functional equivalents of these, but all other terminator sequences that are functionally active in plants are also suitable.

Since plant gene expression is very often not limited to the transcriptional level, a plant expression cassette preferably comprises other sequences that are operably linked, such as translation enhancers, for example, the overdrive sequence, which highlights the 5'-non-translated leader sequence of tobacco mosaic virus, which increases the protein / RNA ratio (Gallie et al., 1987, Nucl. Acids Research 15: 8693-8711).

As described above, plant gene expression must be operably linked with a suitable promoter that activates gene expression with the correct timing or in a cell or tissue specific manner. Usable promoters are constitutive promoters (Benfey et al., EMBO J. 8 (1989) 2195-2202), such as those derived from plant viruses, such as 35S CaMV (Franck et al., Cell 21 (1980)). 285-294), 19S CaMV (also see US 5352605 and WO 84/02913), or plant promoters, such as the Rubisco subunit promoter, which is described in US Patent 4,962,028.

Other preferred sequences for use in operable binding in plant gene expression cassettes are targeting sequences, which are required to guide the gene product into its corresponding cell compartment (see a review in Kermode, Crit. Rev. Plant Sci. 15, 4 (1996) 285-423 and references cited herein), for example, in the vacuole, nucleus, all types of plastids, such as amyloplasts, chloroplasts, chromoplasts, the extracellular space, mitochondria, the endothelial reticulum. plasmid, elaioplasts, peroxisomes and other plant cell compartments.

As described above, plant gene expression can also be obtained by a chemically inducible promoter (see review in Gatz 1997, Annu. Rev. Plant Physiol. Plant Mol. Biol., 48: 89-108). Chemically inducible promoters are particularly suitable when gene expression is desired to occur in a specific time manner. Examples of such promoters are a salicylic acid inducible promoter (WO 95/19443), a tetracycline inducible promoter (Gatz et al. (1992) Plant J. 2,397-404) and an ethanol inducible promoter.

Promoters that respond to biotic or abiotic stress conditions are also suitable, for example, the pathogen-induced PRP1 gene promoter (Ward et al., Plant. Mol. Biol. 22 (1993) 361366), the hsp80 tomato promoter of heat-inducible tomato (US 5,187,267), the potato-inducible alpha-amylase promoter (WO 96/12814) or the wound-inducible pin11 promoter (EP-A-0 375 091).

Especially preferred are those promoters that cause gene expression in tissues and organs in which biosynthesis of fatty acids, lipids and oils occurs in seed cells such as endosperm and developing embryo cells. Suitable promoters are the rapeseed oil napin promoter (US 5,608,152), the Vicia faba USP promoter (Baeumlein et al., Mol Gen Genet, 1991,225 (3): 459-67), the promoter. Arabidopsis (WO 98/45461), the Phaseolus vulgaris phaseoline promoter (US 5,504,200), the Bce4 Brassi-ca promoter (WO 91/13980) or the B4 legumin promoter (LeB4; Baeumlein and others). , 1992, Plant Journal, 2 (2): 233-9), and promoters that elicit seed specific expression in monocotyledonous plants such as maize, barley, wheat, rye, rice and the like. Suitable notable promoters are the Ipt2 or Ipt1 barley gene promoter (WO 95/15389 and WO 95/23230) or barley protein gene promoters, rice glutelin gene, rice orizine gene, rice prolamine, the wheat gliadin gene, the wheat glutelin gene, the corn zein gene, the oat glutelin gene, the Sorghum casirin gene, or the rye secaline gene that are described in WO 99/16890.

As described above, it may be advantageous to include in an expression cassette nucleic acid coding enzymes capable of converting ARA into unsaturated A3 fatty acids such as EPA or DHA. Thus, for example, the expression cassette may also include nucleic acid encoding a Δδ-elongase, oo3 desaturase and / or Δ4 desaturase. Such expression cassettes may be introduced by simultaneously transforming a plurality of individual expression constructs or, preferably, by combining a plurality of expression cassettes into one construct. In addition, a plurality of vectors may be transformed with, in each case, a plurality of expression cassettes and then transferred into the host cell.

Other promoters that are equally especially suitable are those which perform plastid-specific expression, since plastids constitute the compartment in which precursors and some end products are synthesized from lipid biosynthesis. Suitable promoters such as the viral RNA polymerase promoter are described in WO 95/16783 and WO 97/06250, and the Arabidopsis cIpP promoter described in WO 99/46394. Vector DNA can be introduced into prokaryotic and eukaryotic cells by conventional transformation or transfection techniques. The terms "transformation" and "transfection", conjugation and transduction, as used herein, are intended to encompass a multitude of methods known in the prior art by introducing foreign nucleic acid (e.g., DNA) into a host cell. , including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, natural competence, chemically mediated transfer, electroporation or particle bombardment. Suitable methods for transformation or transfection of host cells, including plant cells, can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual., 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and other laboratory books such as Methods in Molecular Biology, 1995, Vol. 44, Agrobacterium protocols, Ed .: Gartland and Davey, Humana Press, Totowa, New Jersey.

In another aspect of the invention there is provided a transgenic non-human organism comprising at least one nucleic acid, gene construct or vector according to an earlier aspect of the invention. The transgenic nonhuman organism can be a microorganism, a nonhuman animal or a plant.

Host cells that are, in principle, suitable for absorbing nucleic acid according to the invention, the gene product according to the invention or the vector according to the invention are all prokaryotic or eukaryotic organisms. The host organisms that are advantageously used are microorganisms such as fungi or yeast, or plant cells, preferably plants or parts thereof. Fungi, yeast or plants are preferably used, especially plants, for example plants such as oil crops, which are high in lipid compounds such as rapeseed oil, evening primrose, flax, thistle, peanuts, oil. rapeseed, flaxseed, soybeans, safflower, sunflower, borage, or plants such as corn, wheat, rye, oats, triticale, rice, barley, cotton, cassava, pepper, Tagetes, Solanacea plants such as potato, tobacco, eggplant and tomato , Vicia species, peas, alfalfa, enclosed plants (coffee, cocoa, tea), Salix species, trees (palm oil, coconut), and perennial grass and forage crops. Especially preferred plants according to the invention are oil crops such as soybean, peanut, rapeseed oil, rapeseed oil, flaxseed, flax, evening primrose, sunflower, safflower, trees (palm oil, coconut).

In an advantageous embodiment, the term "nucleic acid (molecule)" as used herein further comprises the untranslated sequence at the 3 'and 5' ends of the coding gene region: at least 500, preferably 200, especially preferably 100 nucleotides. of the sequence downstream of the 5 'end of the coding region and at least 100, preferably 50, especially preferably 20 nucleotides of the sequence downstream of the 3' end of the coding gene region. An "isolated" nucleic acid molecule is separated from other nucleic acid molecules that are present in the natural source of nucleic acid. An "isolated" nucleic acid preferably has no sequence that naturally flanks the nucleic acid in the genomic DNA of the organism from which the nucleic acid is derived (for example, sequences that are located at the 5 'and 3' ends of the nucleic acid). In various embodiments, the Δθ-elongase, Δ8-desaturase or isolated Δδ-desaturase molecule may comprise, for example, less than approximately 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0 , 1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid is derived. The same applies to other nucleic acid sequences that may be included in an expression cassette, for example sequences encoding a Δδ-elongase, co3 desaturase and / or A4 desaturase. The nucleic acid molecules of the present invention, for example , a nucleic acid molecule with a nucleotide sequence of SEQ ID NO: 1 or residues 7668 to 12077 thereof, or parts of SEQ ID NO: 1 specified by the second to fourth aspects of the invention, may be isolated using standard molecular techniques. and the sequence information provided therein. In addition, for example, a homologous sequence or conserved homologous sequence regions can be identified at the amino acid or DNA level with the help of comparative algorithms. They can be used as a hybridization probe and standard hybridization techniques (such as, for example, those described in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2- ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) to isolate additional nucleic acid sequences that can be used in the process.

In addition, a Perkinsus mari-nus nucleic acid molecule comprising a complete sequence of SEQ ID NO: 1 or a portion thereof may be isolated by polymerase chain reaction, where oligonucleotide primers are used on the basis of this sequence. sequence or parts thereof (for example, a nucleic acid molecule comprising the complete sequence or part thereof may be isolated by polymerase chain reaction using oligonucleotide primers that were generated based on that sequence). For example, mRNA can be isolated from cells (e.g., by the Chirgwin et al. (1979) guanidine thiocyanate extraction method (1979) Biochemistry 18: 5294-5299) and cDNA by reverse transcriptase (e.g. reverse transcriptase Moloney MLV, available from Gibco / BRL, Bethes-da, MD, or AMV reverse transcriptase, available from Seikagaku America, Inc., St.Petersburg, FL).

Synthetic oligonucleotide primers for polymerase chain reaction amplification can be generated based on one of the sequences shown in SEQ ID NO: 1 or with the help of the amino acid sequences detailed in SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4. Particularly suitable primers are shown in the Examples as SEQ ID NO: 5 and SEQ ID NO: 6.

A nucleic acid according to the invention may be amplified by standard PCR amplification techniques using cDNA or, alternatively, genomic DNA as a template (SEQ ID NO 9) and suitable oligonucleotide primers (SEQ ID NO: 5 and SEQ ID NO). : 6). The nucleic acid amplified in this way can be cloned from into a suitable vector and can be characterized by DNA sequence analysis. Oligonucleotides corresponding to a desaturase or elongase nucleotide sequence can be generated by standard synthetic methods, for example, using an automated DNA synthesizer.

The aforementioned nucleic acids and protein molecules with A9-elongase, Δδ-desaturase and / or Δδ-desaturase activity can be used in a process for the production of lino-lactic acid ARA in transgenic organisms.

Then, in another aspect of the invention, there is provided a process for converting linoleic acid or a derivative thereof to arachidonic acid or a derivative thereof into an organism, the process comprising introducing into an organism comprising linoleic acid at least one sequence. nucleic acid comprising: (a) SEQ ID NO: 1 (complete sequence 1047306867), sequence comprising nucleic acid residues 7668 to 12077 of SEQ ID NO: 1 or a homologue thereof; (b) nucleic acid sequences hybridizing under stringent conditions to a nucleic acid sequence of SEQ ID NO: 1 or a sequence comprising nucleic acid residues 7668 to 12077 of SEQ ID NO: 1; c) an isolated nucleic acid sequence encoding polypeptides with A9-elongase, A8-desaturase and Δδ-desaturase activity, wherein the polypeptides are selected from the group consisting of SEQ ID NOS 2, 3 and 4; d) a derivative of a nucleic acid sequence of SEQ ID NO: 1 encoding polypeptides of at least 40% amino acid level identity with SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4; wherein said polypeptides have A9-elongase, Δδ desaturase and Δδ desaturase activity. and expressing said nucleic acid sequence.

In the context of the present invention, a linoleic or arachidonic acid "derivative" is a compound in which the OH of the carboxylic acid moiety is replaced by a moiety of R1, wherein: R1 is coenzyme A (thioester), lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, smooth - difosfatidiIglicerol, lysophosphatidylserine, lysophosphatidylinositol, sphingo base or a radical of the formula II <iL) in which R 2 = hydrogen, lisofosfatidila choline, lysophosphatidylethanolamine, lysophosphatidylglycerol, lisodifosfatidilglcerol, lysophosphatidylserine, lysophosphatidylinositol or C2 Saturated or unsaturated C24-alkylcarbonyl, R3 = hydrogen, saturated or unsaturated C2-C24-alkylcarbonyl, or R2 and R3 are independently of each other a radical of formula Ia: (Ia) wherein n = 2, 3, 4, 5, 6, 7 or 9, m = 2, 3, 4, 5 or 6 and p = 0 or 3; and wherein an oxygen in the radical R1 may be substituted sulfur such that R1 is bound to the remainder of the molecule by a thioester bond.

The processes according to the invention preferably produce total ARA in a content of at least 1 wt.%, Advantageously at least 3 wt.%, Based on total fatty acids in transgenic organisms, preferably in a transgenic plant.

Since a plurality of reaction steps are performed by the starting compounds, the linoleic acid of (18: 2Δ9,12) in the process according to the invention, ARA (20: 4Δ5,8,11 · 14) is not obtained. as a pure product; Minor traits of precursors are always present in the final product. Chemically pure ARA can also be synthesized by the process described above. To this end, ARA or a derivative thereof is isolated from organisms, such as microorganisms or plants or the culture medium within or on which the organisms were grown, or from the organism and culture medium, in the manner known as for example by extraction, distillation, crystallization, chromatography or a combination of these methods. These ARA or ARA derivatives are chemically advantageous for applications in the food industry, cosmetic sector and especially in the pharmaceutical industry sector. The process may include additional steps of converting ARA into a ω-3 fatty acid by introducing nucleic acid encoding a ω-3 desaturase and optionally an A5-elongase and / or Δ4-elongase and / or A4. desaturase.

In another aspect the invention comprises a process for converting 18: 2Δ9,12 (linoleic acid) to 20: 2Δ11,1u, the process comprising introducing into an organism comprising linoleic acid at least one nucleic acid sequence encoding a A9-elongase activity polypeptide comprising: a) a sequence comprising nucleic acid residues 7668 to 9200 of SEQ ID NO: 1, SEQ ID NO: 9 or a homologue thereof; (b) a nucleic acid sequence hybridizing under stringent conditions to a nucleic acid sequence comprising residues 7668 to 9200 of SEQ ID NO: 1 or SEQ ID NO: 9; c) an isolated nucleic acid sequence encoding an A9-elongase activity polypeptide, wherein the polypeptide comprises SEQ ID NO: 2 or SEQ ID NO: 10; d) a derivative of a sequence comprising nucleic acid residues 7668 to 9200 of SEQ ID NO: 1 or SEQ ID NO: 9 encoding a polypeptide of at least 40% amino acid level identity with SEQ ID NO : 2 or SEQ ID NO: 10; wherein said polypeptide has A9-elongase activity; and expressing said nucleic acid sequence.

In another aspect of the invention there is provided a process for converting 20: 2Δ11,14 to 20: 3Δ8,11,14, the process comprising introducing into an organism comprising 20: 2Δ11,14, or comprising linoleic acid and a Δ9 elongase, an isolated nucleic acid sequence encoding an A8-desaturase polypeptide selected from the group consisting of: a) a sequence comprising nucleic acid residues 9351 to 10724 of SEQ ID NO: 1 or a counterpart thereof; (b) nucleic acid sequences hybridizing under stringent conditions to a nucleic acid sequence comprising residues 9351 to 10724 of SEQ ID NO: 1; c) an isolated nucleic acid sequence encoding polypeptides with Δδ desaturase activity, wherein the polypeptide comprises SEQ ID NO: 3; d) a derivative of a sequence comprising nucleic acid residues 9351 to 10724 of SEQ ID NO: 1 encoding a polypeptide with at least 40% amino acid level identity to SEQ ID NO: 3; wherein said polypeptide has A8 desaturase activity; and expressing said nucleic acid sequence.

In another aspect of the invention there is provided a process for converting 20: 3Δ8,11,14 to 20: 4Δ5,8 · 11.14 (ARA), the process comprising introducing into an organism comprising 20: 3Δ8'11 '14 or comprising 20: 2δ11, 14 and an Δδ desaturase, or comprising linoleic acid, an A9 elongase and an Δδ desaturase, an isolated nucleic acid sequence encoding a polypeptide with Δδ desaturase activity and is selected from the group consisting of: a) a sequence comprising nucleic acid residues 10842 to 12077 of SEQ ID NO: 1 or a homologue thereof; (b) nucleic acid sequences hybridizing under stringent conditions to a nucleic acid sequence comprising residues 10842 to 12077 of SEQ ID NO: 1; c) an isolated nucleic acid sequence encoding polypeptides with Δδ desaturase activity, wherein the polypeptide comprises SEQ ID NO: 4; d) a derivative of a sequence comprising nucleic acid residues 10842 to 12077 of SEQ ID NO: 1 encoding a polypeptide of at least 40% identity at the amino acid level of SEQ ID NO: 4; wherein said polypeptide has Δδ desaturase activity. and expressing said nucleic acid sequence. The process may include additional steps for converting ARA into a ω-3 fatty acid by introducing nucleic acid encoding a ω-3 desaturase into the body and optionally a Δδ-elongase and / or a Δ4-elongase and / or an A4- desaturase.

For the processes presented above, it was found that expression was most effectively obtained using galactose induction.

Suitable organisms for production in the process according to the invention are in principle any organism such as microorganisms, non-human animals or plants.

Plants that are suitable are in principle all those plants that are capable of synthesizing fatty acids, such as all dicotyledonous or monocotyledonous plants, algae or moss. Advantageous plants are selected from the group of plant families Adelotheciaceae, A-nacardiaceae, Asteraceae, Apiaceae, Betulaceae, Boraginaceae, Brassica-ceae, Bromeliaceae, Caricaceae, Cannabaceae, Convolvulaceae, Chenopo-diaceae, Cryptheceuceae, Eleptheaeeaeeae Euphorbiaceae, Fabaceae, Geraniaceae, Gramineae, Juglanda-ceae, Lauraceae, Leguminosae, Linaceae, Euglenaceae, Prasinophyceae or vegetable or ornamental plants, such as Tagetes.

Examples that may be mentioned are the following plants selected from the group consisting of: Adelotheciaceae such as the genera Physcomitrella, for example, the genus and species Physcomitrella patens, Anacardiaceae such as the genera Pistacia, Mangifera, Anacardi-um, for example, the genus and species Pistacia vera [pistachio], Mangifer indica [mango] or Anacardium occidentale [cashew], Asteraceae, such as the genera Calendula, Carthamus, Centaurea, Cichorium, Helianthus, Lactuca, Locusta, Tagetes, Valerian, for example. , the genus and species Calendula officinalis [Common Marigold], Carthamus tinctorius [Saffron], Cornflower cyanus [Cornflower], Cichorium intybus [Chicory], Cynara scolymus [Artichoke], Helianthus annus [Sunflower], Lactuca crispa, Lactuca crispa, Lactuca crispa Lac-tuca esculenta, Lactuca scariola, L. ssp. sativa, Lactuca scariola, L. var. grata, Lactuca scariola, L. var. integrifoiia, Lactuca sativa subsp. romana, Locusta communis, Valeriana locusta [salad vegetables], Tagetes lucida, Tagetes erecta or Tagetes tenuifolia [African or trench marigold], Apiaceae, such as the genus Daucus, for example the genus and species Daucus carota [carrot], Betulaceae , such as the genus Corylus, for example, the genera and species Corylus avellana or Corylus colurna [hazelnut], Bo-raginaceae, such as the genus Borago, for example, the genus and species Borago officinalis [borage], such as the genera Brassica, Camelina, Melanosinapis, Sinapis, Arabadopsis, for example, the genera and species Brassica napus, Brassica rapa ssp. [rapeseed oil], Sinapis arvensis, Brassica juncea, Brassica juncea var. juncea, Brassica jun-cea var. crispifolia, Brassica juncea var. folica, Brassica nigra, Brassica si-napioides, Camelina sativa, Melanosinapis communis [mustard], Brassica oleracea [mangold] or Arabidopsis thaliana, Bromeliaceae, such as genera Anana, Bromelia (pineapple), for example, genera and species. Anana comosus, Ananas ananas or Bromelia comosa [pineapple], Ca-ricaceae, such as the genus Carica, such as the genus and species of Carica papaya [papaya], Cannabaceae, such as the genus Cannabis, such as the genus and species of Canabbis sativa [flax], Convolvulaceae, such as the genera Ipomea, Convolvulus, for example, the genera and species Ipomoea batatus, Ipomoea pandurata, Convolvulus potatoes, Convolvulus tiliaceus, Ipomoea fastigiata, Ipomoea tiliacea, Ipomoea triland or Convolvulus pu sweet potato, potato], Chenopodiaceae, such as the genus Beta, as well as the genera and species Beta vulgaris, Beta vulgaris. very high var Beta vulgaris var. vulgaris, Marine Beta, Beta vulgaris var. perennis, Beta vulgaris var. conditiva or Beta vulgaris var. esculenta [sweet beet], Crypthe-codiniaceae, such as genus Crypthecodinium, for example, genus and species Cryptecodinium cohnii, Cucurbitaceae, such as genus Cucurbita, for example, genera and species Cucurbita maxima, Cucurbita mixta, Cucurbita pepo or Cucurbita moschata [pumpkin], Cymbellaceae, such as the genera Amphora, Cymbella, Okedenia, Phaeodactylum, Reimeria, for example, the genus and species Phaeodactylum tricornutum, Ditrichaceae, such as the genera Ditrichaceae, Cerumis, Chrysanthemum, Ceromiis, Distichium, Eccremidium, Lophidion, Philibertiella, Pleuridium, Sa-elania, Trichodon, Skottsbergia, for example the genera and species Ceratodon antarcticus, Ceratodon colophyia, Ceratodon purpurascens, Ceratodon purpureus, Ceratodon purpureus, Ceratodon purpureus. Convolutus Ceratodon purpureus ssp. stenocarpus, Ceratodon purpureus var. Rotundifo-lius, Ceratodon ratodon, Ceratodon stenocarpus, Chrysoblastella chilensis, Ditrichum ambiguum, Ditrichum brevisetum, Ditrichum crispatissimum, Ditrichum falcifolium, Ditrichum flexicaule, Ditrichum flexumule, Ditrichum flexumule, Ditrichum ambumum Ditrichum pallidum, Ditrichum punctulatum, Ditrichum pusillum, Ditrichum pusillum var. Tortile, Ditrichum rhynchostegium, Ditrichum schimperi, Ditrichum tortile, Distichium capillaceum, Distichium ha-genii, Distichium inclinatum, Distichium macounii, Eccremidium floridanum, Eccremidium whiteleggei, Lophidion strictus subulatum, Saelania glaucescens, Trichodon borealis, Trichodon cylindricus or Trichodon cylindricus var. oblongus, Elaeagnaceae, such as the genus Elaeagnus, for example, the genus and species Olea euro-paea [olive], Ericaceae, such as the genus Kalmia, for example, the genus and species Kalmia latifolia, Kalmia angustifolia, Kalmia microphylla, Kalmia polypholia, Kalmia occidentalis, Cistus chamaerhodendros or lucid Kalmia [mountain laurel], Euglenaceae, such as the genus Ascoglena, Astasia, Colacium, Cyclidiopsis, Euglena, Euglenopsis, Hyalaphacus, Khawkinea, Lepocinclis, Phacus, Strombomonas, Trachel, e.g. genus and species Euglena gracilis; Euphorbiaceae, such as the genus Manihot, Jani-pha, Jatropha, Ricinus, for example, the genus and species Manihot utilissima, Janipha manihot, Jatropha manihot, Manihot dulcis, Manihot manihot, Manihot melanobasis, Manihot esculenta or Manihot esculenta Ricinus communis [castor oil plant], Fabaceae, such as the genus Pisum, Albizia, Cathormion, Feuillea, Inga, Pithecolobium, Acacia, Mimosa, Medica-jo, Glycine, Dolichos, Phaseolus, soybean, for example, the genus and Species Pisum sativum, Pisum arvense, Pisum humile [pea], Albizia berteriana, Albizia julibrissin, Albizia lebbeck, Acacia berteriana, Acacia littoralis, Albizia berteriana, Cathormion berteriana, Feuillea berteriana, Ingaellith pransobium peacobium, Transverse frogium berterianum, Pseudalbizzia berteriana, Acacia julibrissin, Acacia nemu, Albizia nemu, Feuilleea julibrissin, Mimosa julibrissin, Mimosa speciosa, Serican-rda julibrissin, Acacia lebbeck, A cacia macrophylla, Albizia lebbeck, Feuilleea lebbeck, Mimosa lebbeck, Mimosa speciosa, Medicago sativa, Medicago fal-cata, Medicago var. [alfalfa] Glycine max Soybean dolichos, Glycine gracilis, Glycine hispida, Phaseolus max, Soy max. , Funariace-ae, such as the genus Aphanorrhegma, Entosthodon, Funaria, Physcomitrel-la, Physcomitrium, for example, genus and species Aphanorrhegma saw-tum, Entosthodon attenuatus, Entosthodon bonplan-dii, Entosthodon califomicus, Entosthodon Entosthodon jame-sonii, Entosthodon leibergii, Entosthodon neoscoticus, Entosthodon rubrise-tus, Entosthodon spathulifolius, Entosthodon tucsoni, American Funaria, Bolariai Funaria, California Funaria, Funaria calvescens, Convolaria Funaria, Funaria fungriana, Hyria fungiiana , Funaria hygrometrica var. arctica, Funaria hygrometrica var. calvescens, Funaria hygrometrica var. convolute, Funaria hygrometrica var. muralis, Funaria hygrometrica var. utahensis, Funaria microstoma, Funaria microstoma var. obtusifolia, Funeral muhlenbergii, Funicular orcuttii, Funeral flat-convex, Funeral polaris, Funeral ravenelii, Funeral rubra, Funeral saw, Funaria sonorae, Funaria sublimbatus, Funeral tucsoni, Physcomitrella californica, Physcomitrella patens, Physcomitrella readerum, Physcomitrium australum , Physcomitrium collenchymatum, Physcomitrium coloradense, Physcomitrium cupuliferum, Physcomitrium drummondii, Physcomitrium eurystomum, Physcomitrium flexifolium, Physcomitrium hookeri, Physcomitrium hookeri var. serratum, Physcomitrium immersum, Physcomitrium kellermanii, Physcomitrium megalocarpum, Physcomitrium pyriform, Pyriform Physcomitrium var. serratum, Physcomitrium rufipes, Physcomitrium sandbergii, Physcomitrium subsphaericum, Physcomitrium washíngtoniense, Geraniaceae, such as the genera Pelargonium, Cocos, Oleum, for example, the genera and species Cocos nucifera, Pelargonium grossularioides or Oleum cocoamin [, coco], such as Cocoum gramineae, Coco Sac-charum genus, for example, the genus and species Saccharum officinarum, Juglan-daceae, such as the genera Juglans, Wallia, for example, genera and species Juglans regia, Juglans ailanthifolia, Juglans sieboldiana, Juglans cinerea, Juglans bixbyi, Juglans californica, Juglans hindsii, Juglans intermedia, Juglans jamaicensis, Juglans major, Juglans microcarpa, Juglans nigra or Wallia nigra [walnut], Lauraceae, such as the genera Persea, Laurus, for example, the genus and species Laurus nobilis. ], Persea americana, Persea gratiosa or Persea persea [avocado], Leguminosae, such as the genus Arachis, for example, the genus and species Arachis hypogae a [peanut], Linaceae.tal as the genera Adenolinum, for example, the genera and species Linum usitatissimum, Linum humile, Linum austriacum, Linum bienne, Linum angustifolium, Linum flavum, Linum grandiflorum, Adenolinum grandiflorum, Linum lewisii, Narbonan linum, Perennial linum, Perennial linum var. lewisii, Linum pratense or Linum trigy-num [linseed], Lythrarieae, such as the genus Punica, for example, the genus and species Punica granatum [pomegranate], such as the genus Gossypi-um, for example, genera and Gossypium hirsutum, Gossypium arboreum, Gossypium barbadense, Gossypium herbaceum or Gossypium thurberi [cotton], Marchantiaceae, such as the genus Marchantia, for example, the genera and species Marchantia berteroana, Marchantia foliacea, Marchantia macropora, Musaceae, such as , for example the genera and species Musa nana, Musa acuminata, Musa paradisiac, Musa spp. [banana], Onagraceae, such as genera Camissonia, Oenothera, for example, genera and species Oenothera biennis or Camissonia bre-vipes [evening primrose], Palmae, such as genus Elaeis, for example, genus and species Elaeis guineensis [palm oil], Papaveraceae, such as, for example, the genus Papaver, for example, genera and species Papaver orientale, Papaver rhoeas, Papaver dubium [poppy], Pedatiaceae, such as the genus Sesamum, for example, genus and species Sesamum indicum [sesame], Piperaceae, such as the genus Piper, Artanthe, Pepero-mia, Steffensia, for example, the genera and species Piper aduncum, Piper amalago, Piper auritum, Piper betei, Piper cubeba, Piper longum, Piper nigrum, Piper retrofractum, Artanthe adunca, Artanthe elonga-ta, Peperomia elongata, Piper elongatum, Steffensia elongata [pepper], Po-aceae, such as the genera Hordeum, Secale, Avena, Sorghum, Andropo-gon, Holcus , Panicum, Oryza, Zea [corn], T riticum, for example, the genera and species Hordeum vulgare, Hordeum jubatum, Hordeum murinum, Hordeum secalinum, Hordeum distichon, Hordeum aegiceras, Hordeum hexasti-chon, Hordeum hexastichum, Hordeum sativum, Hordeum secalinum [cerevea] rye], Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida [oats], Sorghum bicolor, Sorghum halepense, Sorghum saccharatum, Sorghum vulgare, Andropogon drummondii, Bicolor Holcus, Sorghum Holcus, Sorghum ae-thiopicum, Sorghum caundrorum, Sorghum docernum Sorghum drumghum, Sorghum cernuuma durra, Sorghum guineen, Sorghum lanceolatum, Sorghum nervosum, Sorghum saccharatum, Sorghum subglabrescens, Sorghum verticilliflorum, Sorghum vulgare, Holcus halepensis, Sorghum miliaceum, Panicum militaceum [Oize maize], Oryza sativa, Oryza rice Zorea [maize] Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum or Triticum vulgare [wheat], Porphyridiaceae, such as the genera Chroo-thece, Flintiella, Petrovanella, Rhodium Vanilla, Porphyros, Rhodella , for example, the genus and species Porphyridium cruentum, Protea-ceae, such as the genus Macadamia, for example, the genus and species Maca-damia intergrifolia [macadam ia], Prasinophyceae, such as the genera Nep-hroselmis, Prasinococcus, Scherffelia, Tetraselmis, Mantoniella, Ostreococ-cus, e.g. -ca, Mantoniella squamata, Ostreococcus tauri, Rubiaceae, such as the genus Coffea, for example, the genera and species Coffea spp., Coffea arabica, Coffea canephora or Coffea liberica, such as the genus Verbascum, for example, genera and species Verbascum blattaria, Ver-bascum chaixii, Verbascum densiflorum, Verbascum lagurus, Verbascum longifolium, Verbascum lychnitis, Verbascum nigrum, Verbascum phlomoides, Verbascum phoenicum, Verbascum verbascum, Verbascum lympnum, Verbascum olificum -sicum, Nicotiana, Solanum, Lycopersicon, for example, the genera and species Capsicum annuum, Capsicum annuum var. glabriusculum, Capsicum frutescens [pepper], Capsicum annuum [paprika], Nicotiana tabacum, Nicotiana alata, Nicotiana attenuata, Nicotiana glauca, Nicotiana langsdorffii, Nicotiana obtusifolia, Nicotiana quadrivalvis, Nicotiana repanda, Nicotiana rusticus, Nicotiana rusticum [Nicotiana syrup] potato], Solanum me-longena [eggplant], Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersicon Pyriforme, Solanum integrifolium or Solanum lycopersicum [tomato], such as the genus Theobroma, for example, the genus and species Theobroma or cocoa [cocoa] Theaceae, such as the genus Camellia, for example, the genus and species of Camellia sinensis [tea].

Advantageous microorganisms are, for example, fungi selected from the group of families Chaetomiaceae, Choanephoraceae, Crypto-coccaceae, Cunninghameliaceae, Demetiaceae, Moniliaceae, Mortierellace-ae, Mucoraceae, Pythiaceae, Sacharomycetaceae, Saprolegniaceae or Schpracegneae.

Examples of microorganisms that may be mentioned are those of the groups: Choanephoraceae, such as the genera Blakeslea, Choanephora, for example, genera and species Blakeslea trispora, Choanephora cucurbitarum, Choanephora infundibulifera var. cucurbitarum, Mor-tierellaceae, such as the genus Mortierella, for example, the genera and species Mortierella isabellina, Mortierella polycephala, Mortierella ramanniana, Mortierella vinacea, Mortierella zonata, Pythiaceae, such as the genera, Phytium, Phytophthora, for example, the genera and Pythium species debar-yanum, Pythium intermedium, Pythium irregulare, Pythium megalacanthum, Pythium paroecandrum, Pythium sylvaticum, Pythium ultimum, Phytophthora cactorum, Phytophthora cinnamomi, Phytophthora citricola, Phytophthora citrophthora, Phytophthora cryptogea, Phytophthora drechsleri, erythroseptica Phytophthora Phytophthora lateralis, Phytophthora megasperma, Phytophthora nicotianae, Phytophthora nicotianae var. parasitic, Phytophthora pal-mivora, Phytophthora parasitica, Phytophthora syringae, Saccharomycetace-ae, such as the genera Hansenula, Pichia, Saccharomyces, Saccharomy-codes, Yarrowia, for example, the genera and species Hansenula anomalous, Hansenula canadornensis, Hansenula canadornensis, Hansenula canadornensis, capsulata, Hansenula ciferrii, Hansenula glucozyma, Hansenula henricii, Hansenula holstii, Hansenula minuta, Hansenula nonfermentans, Hansenula philodendri, Hansenula polymorpha, Hansenula saturnus, Hansenula subpelliculosa, Hansenula wickerhamii, Pichia angora wingei, Pichia angora, Pichia angora Pichia burtonii, Pichia canadensis, Pichia capsulata, Pichia carsonii, Pichia cellobiosa, Pichia ciferrii, Pichia farinosa, Pichia finans, Pichia glucozyma, Pichia guilliermondii, Pichia haplophila, Pichia henricii membrane , Pichia methanolica, Pichia minuta var. Minute, Pichia Minute var. nonfermentans, Pichia norvegensis, Pichia ohmeri, Pichia pastoris, Pichia philodendri, Pichia pini, Pichia polymorpha, Pichia quercuum, Pichia rhodanensis, Pichia storgitis, Pichia strasburgen-sis, Pichia subpellich Pichia trichia subpellia xylosa, Saccharomyces aceti, Saccharomyces bailii, Saccharomyces bayanus, Saccharomyces bisporus, Saccharomyces capensis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces cerevisiae var. ellipsoideus, Saccharomyces chevalieri, Saccharomyces delbrueckii, Saccharomyces diastaticus, Saccharomyces drosophilarum, Saccharomyces elegans, Saccharomyces ellipsoideus, Saccharomyces fermentati, Saccharomyces Florentinus fragilis Saccharomyces, Saccharomyces heterogenicus Saccharomyces hienipiensis Saccharomyces inusitatus, Saccharomyces italicus, Saccharomyces kluyveri, Saccharomyces krusei, Saccharomyces lactis, Saccharomyces marxianus, Saccharomyces microellipsoides, Saccharomyces montanus, Saccharomyces norbensis, Saccharomyces oleaceus, Paradoxus, Saccharomyces, Saccharomyces pastorianus. For example, the species Schizosaccharomyces japonicus var. japonicus, Schizosaccharomyces japonicus var. versatilis, Schizosaccharomyces malidevorans, Schizosaccha-romyces octosporus, Schizosaccharomyces pombe var. malidevorans, Schi-zosaccharomyces pombe var. pombe, Thraustochytriaceae such as Althornia genera Aplanochytrium, Japonochytrium, Schizochytrium, Thraustochytrium, for example Schizochytrium species aggregatum, Schizochytrium limacinum, Schizochytrium, mangrove, Schizochytrium minutum, Schizochytrium octosporum, Thraustochytrium aggregatum, Thraustochytrium amoeboídeum, Thraustochytrium antacticum, Thraustochytrium arudimenta-le, Thraustochytrium aureum, Thraustochytrium benthicola, Thraustochytrium globosum, Thraustochytrium indicum, Thraustochytrium kerguelense, Thraustochytrium kinnei, Thraustochytrium motivum, Thraustochytrium multi-rudimentale, Thraustochytrium pachydermum, Thraustochytrium proliferum, Thraustochytrium roseum, Thraustochytrium rossii, Thraustochytrium striatum or Thraustochytrium visurgense.

Other advantageous microorganisms are, for example, bacteria selected from the Bacillaceae, Enterobacteriacae or Rhi-zobiaceae families.

Examples that may be mentioned are the following microorganisms selected from the group consisting of: Bacillaceae, such as Bacillus genus, for example, Bacillus acidocaldarius genera and species, Bacillus acidoterrestris, Bacillus alcalophilus, Bacillus amylo-liquefaciens, Bacillus amylolyticus , Bacillus brevis, Bacillus cereus, Bacillus circulans, Bacillus coagulans, Bacillus sphaericus subsp, fusiformis, Bacillus galactophilus, Bacillus globisporus, Bacillus globisporus subsp. marinus, Bacillus halophilus, Bacillus lentimorbus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus polymyxa, Bacillus psychrosaccharolyticus, Bacillus pumilus, Bacillus sphaericus, Bacillus subtilis. subsp. spizizenii, Bacillus subsp subtilis. subtilis or Bacillus thuringiensis; Enterobacteriacae such as the genera Citrobacter, Edwardsiella, Enterobacter, Erwinia, Escherichia, Klebsiella, Salmonella or Serratia, for example, the genera and species Citrobacter amalonaticus, Citrobacter freundii, Citrobacter genomospecies, Citrobacter gillenium, Citrobacter intermediate, Citrobacter gillenii, Citrobacter , Citrobacter murliniae, Citrobacter sp., Edwardsiella hoshinae, Edwardsiella ictaluri, Edwardsiella tarda, Erwinia alni, Erwinia amylovora, Erwinia ananatis, Erwinia billingiae, Erwinia cacticida, Erwinia cancerogena Erainia carninia, Erwinia carnogena, Erainia carninia atroseptica, Erwinia carotovora subsp. betavasculorum, Erwinia carotovora subsp. odori-fera, Erwinia carotovora subsp. wasabiae, Erwinia chrysanthemi, Erwinia cy-pripedii, Erwinia dissolvens, Erwinia herbicola, Erwinia mallotivora, Erwinia milletiae, Erwinia nighfluens, Erwinia persicina, Erwinia psidii, Erwinia pyrifoliae, Erwinia psifii, Erwinia pyrifoliae stewartii, Erwinia tracheiphila, Erwinia uredovora, Escherichia adecarboxylata, Escherichia anindolica, Escherichia aurescens, Escherichia blattae, Escherichia coli, Escherichia coli var. such as Escherichia coli-mutabile, Escherichia fergusonii, Escherichia her-mannii, Escherichia sp., Escherichia vulneris, Klebsiella aerogenes, Klebsiella edwardsii subsp. atlantae, Klebsiella ornithinolytica, Klebsiella oxytoca, Klebsiella planticola, Klebsiella pneumoniae, Klebsiella pneumoniae subsp. pneumoniae, Klebsiella sp., Klebsiella terrigena, Klebsiella trevisanii, Salmonella abony, Salmonella arizonae, Salmonella bongori, Salmonella cholerae-suis subsp. arizonae, Salmonella choleraesuis subsp. bongori, Salmonella choleraesuis subsp. cholereasuis, Salmonella choleraesuis subsp. diarizone-e, Salmonella choleraesuis subsp. houtenae, Salmonella choleraesuis subsp. indica, Salmonella choleraesuis subsp. salamae, Salmonella daressalaam, Salmonella enterica subsp. houtenae, Salmonella enterica subsp. salamae, Salmonella enteritidis, Salmonella gallinarum, Salmonella heidelberg, Salmonella panama, Salmonella senftenberg, Salmonella typhimurium, Serratia en-tomophila, Serratia ficaria, Serratia grimesii, Serratia lique-faciens, Serratia marcescens subs, Serratia marcescens subs. marcescens, Serratia marinorubra, Serratia odorifera, Serratia plymouthensis, Serratia piymu-thica, Serratia proteamaculans, Serratia proteamaculans subsp. quinovora, Serratia quinivorans or Serratia rubidaea; Rhizobiaceae, such as the genera Agrobacterium, Carbophilus, Cheiatobacter, Ensifer, Rhizobium, Sinorhi-zobium, for example, the genera and species Agrobacterium atlanticum, Agrobacterium ferrugineum, Agrobacterium gelatinovorum, Agrobacterium lar-rymoorei, Agrobacterium meteorob, Agrobacterium meteorobium, Agrobacterium rubi, Agrobacterium stellulatum, Agrobacterium tumefaciens, Agrobacterium vitis, Carbophilus carboxidus, Cheiatobacter heintzii, Ensifer adhaerens, Ensifer fredii, Ensifer kostiensis, Ensifer kummerowiae, Ensifer medi, Ensifer meliiceum , Rhizobium etli, Rhizobium fredii, Rhizobium galegae, Rhizobium gallicum, Rhizobium giardinii, Rhizobium hainanense, Rhizobium huakuif, Rhizobium indigoferae, Rhizobium indigoferae, Rhizobium japonicum, Rhizobium legumizobium Rhizobium rhizobium iloti, Rhizobium mongolense, Rhizobium phaseoli, Rhizobium radiobacter, Rhizobium rhizogenes, Rhizobium ruby, Rhizobium sullae, Rhizobium tianshanense, Rhizobium tropici, Rhizobium undicola, Rhizobium vitis, Sinerizium biori, Sinizium rhizobis kummerowiae, Sinorhizobium medicae, Sinorhizobium meliloti, Sinorhizobium morelense, Sinorhizobium saheli or Sinorhizobium xinjiangense.

Other examples of microorganisms advantageous to the process according to the invention are protists or diatoms selected from the Dinophyceae, Turaniellidae or Oxytrichidae family, such as genera and species: Crypthecodinium cohnii, Phaeodactylum tricornutum, Stylonychia mytilus, Stylonychia pchiarata, Stylonychia pustulata, Stylonychia notophora, Stylonychia sp., Colpidium campylum or Colpidium sp.

Those which are advantageously applied in the process according to the invention are transgenic organisms such as fungi such as mortierella or thraustrochytrium, yeasts such as Saccharomyces or Schizosaccharomyces, mosses such as Physcomitrella or Ceratodon, non-human animals such as Caenorhabditis, algae such as Nephrosel-mis, Pseudoscourfielda, Prasinococcus, Scherffelia, Tetraselmis, Mantoniel-la, Ostreococcus, Crypthecodinium or Phaeodactylum or plants such as dicotyledonous or monocotyledonous plants. Organisms which are especially advantageously used in the process according to the invention are organisms which belong to oil producing organisms, that is to say they are used for oil production, such as fungi such as Mortierella or Thraustochytrium, algae such as Nephroselmis, Pseudoscourfielda, Prasinococcus, Scherffelia, Tetraselmis, Mantoniella, Ostreococcus, Crypthecodinium, Phaeodactylum, or plants, in particular plants, preferably oil seed or oil crop plants comprising large amounts of lipid compounds, such as peanut, seed oil rapeseed, rapeseed oil, sunflower, safflower (Carthamus tinctoria), poppy, mustard, flax, castor oil plant, olive, sesame, Ca-calendula, Punica, evening primrose, verbascum, thistle, wild roses, hazelnut, almond, macadamia nuts, avocado, bay, pumpkin, flaxseed, soybeans, pistachios, blot, trees (palm oil, coconut or walnut) or cultivable crops is such as corn, wheat, rye, oats, triticale, rice, barley, cotton, cassava, pepper, Tagetes, Solanaceae plants, such as potato, tobacco, eggplant and tomato, Vicia species, peas, alfalfa or closed plants (coffee , cocoa, tea), Salix species, and perennial grams and forage crops. Preferred plants according to the invention are oil harvesting plants such as peanut, rapeseed oil, rapeseed oil, sunflower, safflower, poppy, mustard, flax, castor oil plant, olive, calendula, punica, Evening primrose, pumpkin, flaxseed, soybean, borage, trees (palm oil, coconut). Especially preferred for the process are plants that have the high content of C18: 2- fatty acids, such as sunflower, safflower, tobacco, verbascum, sesame, cotton, pumpkin, poppy, evening primrose, walnut, flaxseed, flax or thistle. Most especially preferred plants are plants such as safflower, sunflower, poppy, evening primrose, walnut, flaxseed or flax.

It should also be advantageous for the above-described method according to the invention to further introduce into the body other nucleic acids encoding the enzymes of fatty acid or lipid metabolism in addition to the nucleic acids of the first to fourth aspects of the invention.

Such nucleic acids are advantageously derived from plants such as algae, for example, algae of the Prasinophyceae family such as the genera Heteromastix, Mammella, Mantoniella, Micromonas, Nephrosel-mis, Ostreococcus, Prasinococcus, Pasudoscourfielda, Pycimimonas, Pycimimonas Scherffelia or Tetraselmis, such as the genera and species Heteromastix longifillis, Mamiella gilva, Mantoniella squamata, Micromonas pusilla, Nephroselmis pyriformis, Nephroselm rotunda, Ostreococcus tauri, Ostreococcus sp. Prasinocladus Ascus, Prasinocladus lubricus, Pycnococcus evidolii, Pyramimonas amylifera, Pyramimonas disomata, Pyramimonas obovata, Pyramimonas orientalis, Pyramimonas spinifera, Pyramimonas sp. gracilis tetraselmis, tetraselmis hazeni, tetraselmis impellucida, tetraselmis inconspicua, tetraselmis levis, tetraselmis maculata, tetraselmis marina, tetraselmis striata, tetraselmis subcordi-formis, tetraselmis suecica, tetraselmis tetrabrachia, tetraselmis tetrathele, warty tetraselmis, tetraselmis warty fo. rubens or Tetraselmis sp. or algae of the Euglenaceae family such as the genus Ascoglena, Astasia, Colacium, Cyclidiopsis, Euglena, Euglenopsis, Hyalophacus, Khawkinea, Lepocinclis, Phacus, Strombomonas or Trachelomonas, such as the genus and species Euglena accuse, Euglena geniculate, Euglena gril mixoc-ylindracea, Euglena rostrifera, Euglena viridis, Colacium stentorium, Trachelomonas cylindrica or Trachelomonas volvocina. Advantageously used nucleic acids are derived from algae of the genera Euglena, Mantoniella or Ostreococcus.

Other advantageous plants are algae such as Isochrysis or Crypthecodinium, algae / diatoms such as Thalassiosira or Phaeodact-ylum, mosses such as Physcomitrella or Ceratodon, or taller plants such as Primulaceae such as Aleuritia, Calendula stellata, Osteospermum hypersumensus or Osteospermum spinescensium , microorganisms such as fungi such as Aspergillus, Thraustochytrium, Phytophthora, Entomophthora, Mucor or Mortierella, bacteria such as Shewanella, yeast or animals such as nematodes such as Caenorhabditis, insects, frogs, abalone, or fish. The isolated nucleic acid sequences according to the invention are advantageously derived from a vertebrate animal. Preferably, the nucleic acid sequences are derived from the Vertebrata classes; Euteleostomi, Actinopterygii; Neopterygii; Teleostei; Euteleostei, Protacanthopterygii, Salmoniformes; Salmonidae or Oncorhynchus or Vertebrata, Amphibia, Anura, Pipidae, Xenopus or Evertebrata such as Proto-chordata, Tunicata, Holothuroidea, Cionidae such as Amaroucium constella-tum, Botryllus schlosseri, Ciona intestinalis, Molgula citrina viris, Molgula manhat or Perisula manhat Styela leaves. Nucleic acids are especially advantageously derived from fungi, animals, or plants such as algae or moss, preferably of the order Salmoniformes, such as the Salmonidae family, such as the genus Salmo, for example, the genera and species Oncorhynchus mykiss, Trutta trutta or Salmo trutta fario, from algae such as Mantoniella or Ostreococcus or diatoms such as Thalassiosira or Phaeodactylum or algae such as Crypthecodinium.

In a preferred embodiment, the process comprises the step of obtaining an intact cell or organism comprising the nucleic acid sequences used in the process, wherein the cell and / or organism is transformed with a nucleic acid sequence according to the invention which encodes A9-elongase, and / or Δδ desaturase and / or A5-desaturase, a gene construct or a vector as described above, alone or in combination with other nucleic acid sequences encoding the proteins of fatty acid metabolism or lipid. In another preferred embodiment, this process also comprises the step of obtaining free oils, lipids or fatty acids from the organism or culture. The culture may, for example, take the form of a fermentation culture, for example in the case of the cultivation of microorganisms such as, for example, Mortierella, Tha-lassiosira, Mantoniella, Ostreococcus, Saccharomyces or Thraustochytrium, or greenhouse or field growth of a plant. The cell or organism thus produced is advantageously a cell of an oil producing organism, such as an oil crop, such as, for example, peanuts, rapeseed oil, rapeseed oil, flaxseed, flax, peanuts, soybeans, safflower, flax, sunflowers or borage.

In the case of plant cells, plant tissue or plant organs, "growing" is understood to mean, for example, cultivation in or on a nutrient medium, or intact plant on or within a substrate, for example, in a hydroponic crop, composed of plantation or in arable land.

For purposes of the invention, "transgenic" or "recombinant" means with respect to, for example, a nucleic acid sequence, an expression cassette (= gene construct) or a vector comprising the nucleic acid sequence or an organism. transformed with nucleic acid sequences, expression cassettes or vectors according to the invention, all such constructs are performed by recombinant methods in which either a) the nucleic acid sequence according to the invention, or b) a genetic control sequence that is operably linked to the nucleic acid sequence according to the invention, for example, a promoter, or c) a) and b) are not located in their natural genetic environment or have been modified by recombinant methods, i.e. being possible for the modification to take the form of, for example, a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. The natural genetic environment is understood to mean the natural genomic or chromosomal locus in the original organism or presence in a genomic library. In the case of a genomic library, the natural genetic environment of the nucleic acid sequence is preferably retained, at least in part. The environment flanks the nucleic acid sequence at least on one side and has a sequence length of at least 50 bp, preferably at least 500 bp, especially preferably at least 1000 bp, preferably at least 5000 bp. A naturally occurring expression cassette - for example, the naturally occurring combination of the natural promoter of nucleic acid sequences with the corresponding Δ5 desaturase gene - becomes a transgenic expression cassette when this expression cassette is modified by unnatural, synthetic ("artificial") methods such as, for example, mutagenic treatment. Suitable methods are described, for example, in US 5,565,350 or WO 00/15815.

A transgenic organism or transgenic plants for the purposes of the invention is then understood to mean, as above, that the nucleic acids used in the process are not in their natural place in the genome of an organism, and nucleic acids may be expressed homologously or heterologously. . However, as mentioned, transgenic also means that while the nucleic acids according to the invention are in their natural position in the genome of an organism, the sequence has been modified with respect to the natural sequence, and / or that the sequences regulators of natural sequences have been modified. Transgenic is preferably understood to mean expression of nucleic acids according to the invention at an unnatural place in the genome, that is, homologous or, preferably, heterologous expression of nucleic acids occurs. Preferred transgenic organisms are fungi such as Mortierella or Phytophtora, mosses such as Physcomitrella, algae such as Mantoniella, Euglena, Crypthecodinium or Ostreococ-cus, diatoms such as Thalassiosira or Phaeodactylum, or plants such as oil crops.

The host organisms or organisms for the nucleic acids, the expression cassette or the vector used in the process according to the invention are in principle advantageously all organisms which are capable of synthesizing fatty acids, specifically unsaturated fatty acids. and / or which are suitable for expression of recombinant genes. Examples that may be mentioned are plants such as Arabidopsis, Asteraceae such as Calendula or harvesting plants such as soybean, peanut, castor oil plant, sunflower, corn, cotton, flax, rapeseed oil, coconut, palm oil, safflower (Carthamus tinctorius) or cocoa seed, microorganisms such as fungi, for example the genus Mortierella, Thraustochytrium, Saprolegnia, Phytophtora or Pythium, bacteria such as the genus Escherichia or Shewanella, yeasts such as genus Saccharomyces, cyanobacteria, ciliates, algae such as Mantoniella, Euglena, Thalassiosira or Ostreococcus, or protozoa such as dinofla-gellates such as Crypthecodinium. Preferred organisms are those which are naturally capable of synthesizing significant amounts of oil such as fungi such as Mortierella alpina, Pythium insidiosum, Phytophtora infestans, or plants such as soybean, rapeseed oil, coconut, palm oil, safflower. , flax, flax, castor oil plant, calendula, peanut, cocoa or sunflower seed, or yeast, such as Saccharomyces cerevisiae, such as soybean, flax, rapeseed oil, safflower, sunflower, calendula, mortierella or saccharomyces cerevisiae which are especially preferred. In principle, the host organisms are, in addition to the above-mentioned transgenic organisms, also transgenic animals, advantageously non-human animals, for example C. elegans, Ciona intestinalis or Xenopus laevis.

Also useful are host cells detailed in: Goeddel, Gene Expression Technology ·. Methods in Enzymology 185, Academic Press, San Diego, CA (1990). The expression strains that can be used, for example, those with lower protease activity, is described in: Gottesman, Sr, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128 .

These include plant cells and certain plant tissues, organs and parts in all their phenotypic forms such as anthers, fibers, root hair, stems, embryos, stem, cotyledons, petioles, harvested material, plant tissue, reproductive tissue and cultures. which are derived from the current transgenic plant and / or may be used to make the transgenic plant.

Transgenic plants comprising the polyunsaturated fatty acids synthesized in the process according to the invention may be advantageously and directly marketed without any need for synthesized oils, lipids or fatty acids to be isolated. Plants for the process according to the invention are listed as meaning intact plants and all plant parts, plant organs or plant parts such as leaf, stalk, seeds, root, tubers, anthers, fibers, root hair, Stalks, embryos, stem, cotyledons, petioles, harvested material, plant tissue, reproductive tissue, and cell culture that are derived from the current transgenic plant and / or may be used to make the transgenic plant. In this context, the seed comprises all parts of the seed such as seed husk, epidermal cells, seed cells, endosperm or embryonic tissue. However, the compounds produced in the process according to the invention may also be isolated from organisms, advantageously plants, in the form of their oils, fats, lipids and / or free fatty acids. Polyunsaturated fatty acids produced by this process can be obtained by harvesting the organisms, or from the crop on which they grow, or from the field. This can be done by pressing or extracting plant parts, preferably plant seeds. In this context, oils, fats, lipids and / or free fatty acids may be obtained by what is known as cold beating or cold pressing without applying heat. In order to facilitate breakability of plant parts, specifically seeds, they are pre-broken, steamed or roasted. Seeds that have been pretreated in this manner can be subsequently squeezed or extracted with solvents such as hot hexane. The solvent is subsequently removed. In the case of microorganisms, the latter are, after harvesting, for example, extracted directly without further processing steps or, after disruption, extracted by various methods with which the skilled worker is familiar. In this way, more than 96% of the compounds produced in the process can be isolated. Thereafter, the resulting products are further processed, i.e. refined. In this process, substances such as plant mucilages and suspended substances are first removed. What is known as degreasing may be carried out enzymatically or, for example, chemically physically by addition of acid such as phosphoric acid. Thereafter, the free fatty acids are removed by treatment with a base, for example sodium hydroxide solution. The resulting product is washed thoroughly with water to remove the alkali remaining in the product and then dried. To remove the pigment that remains in the product, the products are subjected to bleaching, for example, using cargo earth or active charcoal. In the end, the product is deodorized, for example using steam.

Fatty acids produced by the processes of the present invention may be isolated from the body in the form of an oil, lipid or free fatty acid. For example, suitable organisms are those mentioned above. Preferred organisms are transgenic plants.

One embodiment of the invention is then oils, lipids or fatty acids of formula I or fractions thereof which were produced by the process described above, especially preferably oil, lipid or a fatty acid composition comprising a compound of formula I and being derived from transgenic plants. .

Another embodiment according to the invention is the use of oil, lipid, fatty acids and / or fatty acid composition in food, edible, cosmetic or pharmaceutical products. The oils, lipids, fatty acids or fatty acid mixtures according to the invention may be used in the manner in which the skilled worker is familiar with mixing with other animal origin oils, lipids, fatty acids or fatty acid mixtures such as such as fish oils. These oils, lipids, fatty acids or mixtures of fatty acids which are vegetable compounds and animal components may also be used for the preparation of food, edible, cosmetic or pharmacological products. The term "oil", "lipid" or "fat" is understood to mean a fatty acid mixture comprising unsaturated, saturated, preferably esterified fatty acid (s). The oil, lipid or fat is preferably high in free or advantageously esterified polyunsaturated fatty acid (s), in particular linoleic acid, γ-linolenic acid, dihomo-γ-linolenic acid, arachidonic acid, α-linolenic acid, stearidonic acid, eicosatetraenoic acid, eicosapentaenoic acid, docosapentaeenoic acid or docosaexaenoic acid. The amount of unsaturated esterified fatty acids is preferably amounts of approximately 30%, a content of 50% is more preferred, a content of 60%, 70%, 80% or more is even more preferred. For analysis, the fatty acid content may, for example, be determined by gas chromatography after converting the fatty acids into methyl esters by transesterification. The oil, lipid or fat may comprise various other saturated or unsaturated fatty acids, for example calendulic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid and others. The content of the various fatty acids in the oil or fat may vary, in particular depending on the starting organism. The ARA produced in the process may be, as described above, in the form of a fatty acid derivative, for example, sphingolipids, phosphoglycerides, lipids, glycolipids, phospholipids, monoacylglycerol, diacylglycerol, triacylglycerol or other fatty acid esters. ARA and other polyunsaturated fatty acids that are present may be released, for example, by treatment with alkali, for example aqueous KOH or NaOH, or acid hydrolysis, advantageously in the presence of an alcohol such as methanol or ethanol, or by divising. enzymatic, and isolated by, for example, phase separation and subsequent acidification by, for example, H2SO4. Fatty acids can also be released directly without the processing step described above.

Upon introduction into an organism, advantageously a plant or plant cell, the nucleic acids used in the process may either be present in a separate plasmid or advantageously integrated into the host cell genome. In the case of integration into the genome, the integration may be random or may be effected by re-combining such that the native gene is replaced by the introduced copy, whereby the production of the desired compound by the cell is modulated, or by the use of a gene in trans, such that the gene is operably linked with a functional expression unit comprising at least one sequence that guarantees expression of a gene, and at least one sequence that ensures polyadenylation of a functionally transcribed gene. . Nucleic acids are advantageously introduced into organisms by multi-expression cassettes or constructs for multi-parallel expression, advantageously into plants for multiparallel seed specific expression of genes.

If microorganisms such as yeast, such as Saccha-romyces or Schizosaccharomyces, fungi such as Mortierella, Aspergillus, Phytophtora, Entomophthora, Mucor or Thraustochytrium, algae such as Isochrysis, Mantoniella, Euglena, Ostreococche, Phainium-Cryptus or Phainiumodactyl organisms are used In the process according to the invention, these organisms will advantageously be grown in fermentation cultures.

If the microorganisms are used as organisms in the process according to the invention, they are grown or grown in the manner in which the skilled worker is familiar, depending on the host organism. As a rule, microorganisms are grown in a liquid medium comprising a carbon source, usually in the form of sugars, a nitrogen source, usually in the form of organic nitrogen sources such as yeast extract or salts such as sulfate. of ammonium, trace elements such as iron, manganese and magnesium salts and, if appropriate, vitamins, at temperatures of between 0 ° C and 100 ° C, preferably between 10 ° C and 60 ° C, while passing in oxygen. The pH of the liquid medium can either be kept constant, ie regulated during the cultivation period or not. Crops can be grown in batches, semi-split or continuously. Nutrients may be supplied at the beginning of fermentation or may be fed semicontinuously or continuously. The polyunsaturated fatty acids produced may be isolated from the organisms as described above by methods known to the skilled worker, for example by extraction, distillation, crystallization, appropriate salt precipitation, and / or chromatography. To this end, the organisms may advantageously be previously disrupted.

If the host organisms are microorganisms, the process according to the invention is advantageously carried out at a temperature of between 0 ° C and 95 ° C, preferably between 10 ° C and 85 ° C, especially preferably between 15 ° C and 75 ° C. most especially preferably between 15 ° C and 45 ° C.

In this process, the pH value is advantageously maintained between pH 4 and 12, preferably between pH 6 and 9, especially preferably between pH 7 and 8. The process according to the invention may be batch, semi-batch or continuous. An assessment of known cultivation methods can be found in Chmiel's book (Bioprozeptechnik 1. Einführung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or Storhas's book (Bioreaktoren und periphere Einrich-tungen [Bioreactors and peripheral equipment] (Vieweg Verlag, Braunsch-weig / Wiesbaden, 1994)). The culture medium to be used must adequately meet the requirements of the strains in question. Descriptions of the culture media for various microorganisms can be found in the book "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington D. C, USA, 1981).

As described above, these media which may normally be employed in accordance with the invention comprise one or more carbon sources, nitrogen sources, inorganic salts, vitamins and / or trace elements.

Preferred carbon sources are sugars such as mono-, di- or polysaccharides. Examples of very good carbon sources are glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Sugars may also be added to the media by complex compounds such as molasses or other sugar refining by-products. Adding mixtures from a variety of carbon sources can also be advantageous. Other possible carbon sources are oils and fats such as, for example, soybean oil, sunflower oil, peanut oil and / or coconut fat, fatty acids such as, for example, palmitic acid, stearic acid and / or linoic acid, polyalcohols and / or alcohols such as, for example, glycerol, ethanol and / or methanol, and / or organic acids such as, for example, lactic acid and / or acetic acid.

Nitrogen sources are normally organic or inorganic nitrogen compounds or materials comprising these compounds. Examples of nitrogen sources include liquid or gaseous ammonium or ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrate, urea, amino acids or nitrogen sources. complexes such as macerated corn liquor, soy meat, soy protein, yeast extract, meat extract and the like. Nitrogen sources can be used individually or as a mixture.

Inorganic salt compounds which may be present in the media include calcium chloride, phosphorus and sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

Inorganic sulfur-containing compounds such as, for example, sulfates, sulfides, dithionides, tetrationates, thiosulfates, sulfides, or else organic sulfur compounds such as mercaptans and thiols, may be used as sulfur sources for the production of fine chemicals containing sulfur, in particular methionine. Phosphoric acid, potassium hydrogen phosphate or dipotassium hydrogen phosphate or salts containing sodium may be used as sources of phosphorus.

Chelating agents may be added to the medium to keep the metal ions in the solution. Particularly suitable chelating agents include dihydroxyphenols such as catechol or protocatecuate and organic acids such as citric acid.

Fermentation media used according to the invention for cultivating microorganisms usually also comprise other growth factors, for example, such as vitamins or growth promoters which include biotin, riboflavin, thiamine, folic acid, nioquinic acid, pantothenate and pyridoxine. Growth factors and salts are often derived from complex media components such as yeast extract, molasses, macerated corn liquor and the like. Suitable precursors may also be added to the culture medium. The exact composition of the medium compounds depends heavily on particular experience and is decided individually for each specific case. Information on media optimization can be found in the book "Applied Microbiol. Physiology, A Practical Approach" (Editors P. Rhodes, P. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). Growth media may also be obtained from commercial suppliers, for example Standard 1 (Merck) or BHI (Heart Brain Infusion, DIFCO) and the like.

All media components are sterilized either by heat (20 min at 1.5 bar and 121 ° C) or by filter sterilization. The components can either be sterilized together or, if necessary, separately. All components of the medium may be present at the beginning of cultivation or added continuously or in batch as desired. The culture temperature is usually between 15 ° C and 45 ° C, preferably 25 ° C to 40 ° C, and may be kept constant or may be changed during the experiment. The pH of the medium should be in the range of 5 to 8.5, preferably about 7.0. The pH for cultivation can be controlled during cultivation by adding basic compounds such as sodium hydroxide, potassium hydroxide, ammonium and aqueous ammonia or acidic compounds such as phosphoric acid or sulfuric acid. Foaming can be controlled by employing defoamers such as, for example, fatty acid polyglycol esters. To maintain the stability of plasmids, suitable substances which have a selective effect, for example antibiotic, may be added to the medium. Aerobic conditions are maintained by introducing oxygen or oxygen-containing gas mixtures such as, for example, ambient air into the culture. The culture temperature is usually 20 ° to 40 ° C and preferably 25 ° C to 40 ° C. Cultivation is continued until desired product formation is at a maximum. This goal is usually achieved within 10 to 160 hours.

Fermentation broths thus obtained, in particular those containing polyunsaturated fatty acids, usually contain a dry mass of 7.5 to 25% by weight. The fermentation broth can then be processed as well. The biomass may, as required, be completely or partially removed from the fermentation broth by separation methods such as, for example, centrifugation, filtration, decanting or a combination of these methods or left completely in said broth. It is advantageous to process the biomass after its separation.

However, the fermentation broth can also be thickened or concentrated without separating the cells using known methods such as, for example, with the aid of a rotary evaporator, thin film evaporator, reverse osmosis drop film evaporator or through nanofiltration. Finally, this concentrated fermentation broth can be processed to obtain the fatty acids present in it.

The fatty acids obtained in the process are also suitable as a starting material for the chemical synthesis of additional products of interest. For example, they may be used in combination with one another or alone for the preparation of pharmaceuticals, edibles, animal foods or cosmetics. All nucleic acid sequences used in the process according to the invention are advantageously derived from a eukaryotic organism, such as a plant, a microorganism or an animal. Nucleic acid sequences are preferably derived from the order Salmoniformes, algae such as Mantoniella, Crypthecodinium, Eugle-na or Ostreococcus, fungi such as the genus Phytophthora or diatom-ceas such as the genera Thalassiosira or Phaeodactylum. The invention will now be described in greater detail with reference to the following Examples and the drawings in which: Figure 1 shows various synthetic reaction series for the ω-6 and u> -3 fatty acid biosynthesis.

Figure 2 is a trace of gas chromatography showing the conversion of Δ9, 12-18: 2 (linoleic acid) to Δ11, 14-20: 2 by heterologous expression of the P. marinus A9-elongase sequence (SEQ ID NO: 1, residues 7668 to 9200) in yeast or galactose induced (Figure 2A) or glucose (Figure 2B).

Example 1 - Cloning of a Perkinsus marinus FAE1 elonqase Perkinsus marinusi is an oyster protozoan parasite capable of synthesizing saturated and unsaturated fatty acids including essential fatty acid, arachidonic acid [20: 4 (n-6) j. P. marinus employs the delta-8 (Δ -8) desaturase reaction series to synthesize arachidonic acid. Materials and methods.

Growth and harvest of P. marinus Perkinsus marinus meronts were grown at 28 ° C in a medium prepared as described by La Peyre et al. (J Eukaryot Microbiol 1993; 40: 304-10) and contained amino acids, nucleotides, carbohydrate, and vitamins, however no fetal bovine serum.

Nucleic acid manipulation and PCR-based cloning DNA was extracted from cells using a DNeasy DNA minikit (Qiagen). The DNA was amplified with del-ta5 desaturase gene specific primers as follows: reactions were heated at 95 ° C for 2 min followed by 35 cycles at 95 ° C for 1 min, 2 minutes at 52 ° C and 72 ° C for 4 min. minutes, then a single step at 72 ° C for 5 minutes. PCR amplification products were cloned into TOPO vector (Invitro-gen) and verified by sequencing. The FAE elongase gene was amplified with gene-specific primers (Table I) designed for the 5 'and 3' ends of the coding region, with restriction sites to facilitate cloning into the yeast vector (Table I). The forward primers for pYES2 yeast expression vector cloning (Invitrogen) were designed to contain a G / A at -3 position and a G at +4 position to improve translational initiation in eukaryotic cells.

Oligonucleotide primers used in this study Perkinsus marinus transcripts were analyzed by PCR reverse transcriptase (RT - PCR). Total RNA was extracted from cells using an RNeasy (Qiagen) plant minikit. First the strand cDNA was synthesized from total RNA using the SMARTE RACE cDNA Amplification kit (BD-Clontech, Basingstoke, UK) according to the manufacturer's instructions. Single stranded cDNAs were amplified with the following primers.

FAE Front Peron 5 '- GGAATTCGAGGAGTAGGATCTTATCTGAGGATAGTC A-CACTAGTCGTACT-3' FAE Reverse Peron 5'-CATCTGCGAATACTAACCATACATT

Reactions were heated at 95 ° C for 2 minutes followed by 30 cycles at 94 ° C for 30 seconds, 30 seconds at temperatures ranging from 55 to 72 according to the designated primer and 72 ° C for 2 minutes, then a single step at 72 ° C for 10 minutes. PCR amplification products were cloned into TOPO vector (Invitrogen) and verified by sequencing. Surprisingly it has been shown that the transcripts of A9-elongase, Δδ desaturase and Δδ desaturase are all found in the same mRNA. This is the first example showing PUFA genes to be organized into an operon-like structure.

In further investigation the specificity of A9-elongase was analyzed. For this purpose the coding sequence for this gene was amplified by RT-PCR as described above using the following primers.

Elo2For: 5 '- ATGCAAGTTCCCGCGGAGCATCACTCC -3' Elo2Rev: 5 '- CGTT ACGCAT C AAT ATT AT GCAT AGCCAACC -3' The amplified PCR product was then cloned into a pCRscript vector according to the manufacturer's recommendations (Stratagen). In a second PCR step the modified yeast expression sequences were introduced using the following primers.

Yp Expression 5 'Front Elo2 Kpn - TTGGTACCATGGGATTTCCTGCGGAG -3' 5 'Reverse Sac Elo1 - GGG AGCT CTC ACGCAT ATAT AT GCAT AGC-3' The sequence of primers is determined in the 5 'to 3' orientation. Restriction sites used for cloning are in bold. RESULTS

Isolation of P.marinus FAE1 elonqase Using publicly available data derived from a P.marinus genome sequencing project conducted by TIGR (http://www.tigr.org/tdb/e2k1/pmg /) a contig was identified. (1047306867) which showed significant homology to known elongases, with the target sequence (designated Elol Para, SEQ ID NO: 9) consisting of an open reading frame of 511 residues and no introns. The putative amino acid derived from the designated sequence is SEQ ID NO: 10.

Functional Characterization in Saccharomvces cerevisiae The full-size cDNA corresponding to Δ9 putative fatty acid elongase (SEQ ID NO: 9) was cloned into pYES2 yeast expression vector to determine a designated pYPmFAE construct. The S. cerevisiae W303-1A strain was transformed with pYPmFAE or the empty vector as a control. Transformed cells were grown in minimal medium containing raffinose, and were induced with 2% galactose. After 48 hours of growth the total yeast fatty acids were extracted and the resulting FAMEs analyzed by GC. GC analysis (Figure 2) revealed that pYPmFAE-transformed yeast cells produced an additional fatty acid that was identified as eicosadenoic acid indicating that the clone gene encoded a delta 9 elongase fatty acid. The yeast cell expressing P. marinus delta 9 fatty acid elongase is able to recognize the substrate C18: 2 (c9,12) with a conversion rate of 8.2%. Table 1 shows the fatty acid content of yeast cells after transformation with pYPmFAE (+) or empty vector pYES2 (-) and induction with 2% galactose. The conversion percentage at 18: 2δ9,12 to 20: 2δ11,14, for example, is calculated by the equation: Table 1 The results presented in Table 1 show that no elongase activity was detected with 20: 4Δ5,8 '11, 14, and a minimum activity (1% conversion) at 18: 3A6ISI12. Therefore it appears that Δ9 elongase fatty acid is selective for linoleic acid and does not act to prolong other PUFAs.

Claims (5)

Gene construct, characterized in that it comprises a nucleic acid sequence operably linked to one or more heterologous regulatory sequences to the nucleic acid sequence, wherein the nucleic acid sequence encodes a polypeptide with A9-elongase activity. and is selected from the group consisting of: a sequence comprising SEQ ID NO: 9 and degenerate nucleic acid sequences thereof encoding the same amino acid sequence. Vector, characterized in that it comprises a gene construct as defined in claim 1. Transgenic microorganism, characterized in that it comprises a gene construct as defined in claim 1 or a vector as defined in claim 2. Process for the conversion of 18: 2Δ9,12 (linoleic acid) to 20: 2δ11,14, characterized in that it comprises introducing into a microorganism comprising linoleic acid at least one nucleic acid sequence encoding a polypeptide having A9-elongase activity and comprising: a sequence comprising SEQ ID NO: 9 and degenerate nucleic acid sequences thereof encoding the same amino acid sequence; and expressing said nucleic acid sequence. Process according to Claim 4, characterized in that it further includes the galactose induction step.